Chemical synthesis and screening of bicyclic peptide libraries

ABSTRACT

Disclosed herein are bicyclic peptide compounds, compositions comprising same, methods for making same, and libraries comprising same. The disclosed compounds, in various aspects, are useful for treating a variety of disorders, including inflammatory disorders, autoimmune disorders, and disorders of uncontrolled cellular proliferation. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/893,203, filed Nov. 23, 2015, which is a 371 U.S. National Stageof International Application No. PCT/US2014/039332, filed May 23, 2014,which claims the benefit of priority to U.S. Provisional Application No.61/826,805, filed May 23, 2013, each of which are hereby incorporatedherein by reference in their entireties.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numbersGM062820 and CA132855 awarded by the National Institutes of Health. Thegovernment has certain rights in this invention.

BACKGROUND

Protein-protein interactions represent a new class of exciting drugtargets, which are generally considered “undruggable” by conventionalsmall-molecule approaches, because their binding sites are usuallylarge, flat surfaces that lack well defined pockets for small moleculesto bind.

Protein-protein interactions (PPIs) are of central importance inessentially all biochemical pathways, including those involved indisease processes. PPIs therefore represent a large class of new,exciting drug targets (Wells, J and McClendon, C. Nature, 2007, 450,1001-1009). However, PPIs are considered the prototypical “undruggable”or “challenging” targets for the conventional small-molecule approach,because PPIs usually involve large, flat interfaces, with which a smallmolecule generally does not make enough points of contact to impart highaffinity or specificity. On the other hand, it has become relativelystraightforward to develop specific, high-affinity antibodies againstany protein epitopes including flat surfaces. Non-immunoglobulin proteinscaffolds have also been engineered into specific binders to targetproteins through library screening and/or in vitro evolution (Koide, Aet al. J. Mol. Biol., 1998, 284, 1141-1151; Beste, G et al. Proc. Natl.Acad. Sci. USA, 1999, 96, 1898-1903; Xu, L. H. et al. Chem. Biol., 2002,9, 933-942; Rutledge, S E et al. J. Am. Chem. Soc., 2003, 125,14336-14347; Steiner, D et al. J. Mol. Biol., 2008, 382, 1211-1227).These antibody and protein binders possess large binding surfaces oftheir own and are capable of making multiple contacts with a targetsurface (e.g., those involved in PPIs). Unfortunately, protein-baseddrugs are impermeable to the mammalian cell membrane; as such they aregenerally limited to targeting extracellular proteins and are not orallyavailable. Recently, there have been great interests in developingmacrocyclic compounds such as cyclic peptides as PPI inhibitors (Koide,A et al. J. Mol. Biol., 1998, 284, 1141-1151; Beste, G et al. Proc.Natl. Acad. Sci. USA, 1999, 96, 1898-1903; Xu, L. H. et al. Chem. Biol.,2002, 9, 933-942; Rutledge, S E et al. J. Am. Chem. Soc., 2003, 125,14336-14347; Steiner, D et al. J Mol. Biol., 2008, 382, 1211-1227;Dewan, V et al. ACS Chem. Biol., 2012, 7, 761-769; Wu, X et al. Med.Chem. Commun., 2013, 4, 378-382; Tavassoli, A et al. ACS Chem. Biol.,2008, 3, 757-764; Millward, S W et al. ACS Chem. Biol., 2007, 2,625-634; Zhou, H et al. J. Med. Chem., 2013, 56, 1113-1123; Yamagishi, Yet al. Chem. Biol., 2011, 18, 1562-1570; Ardi, V C et al. ACS Chem.Biol., 2011, 6, 1357-1366; Leduc, A M et al. Proc. Natl. Acad. Sci. USA,2003, 100, 11273-11278). These macrocycles typically have molecularweights between 500 and 2000 and occupy a largely untapped therapeuticspace that is often referred to as the “middle space.” Because of theirrelatively large sizes and therefore ability to make multiple points ofcontact with a target, they are able to compete with proteins forbinding to flat surfaces, and yet retain many of the pharmacokineticproperties of small molecules. Bicyclic peptides have also beengenerated in order to further contain their structures and improve theirbinding affinity/specificity and metabolic stability (Sun, Y et al. Org.Lett., 2001, 3, 1681-1684; Virta, P and Lonnberg, H J. J Org. Chem.,2003, 68, 8534; Hennis, C et al. Nat. Chem. Biol., 2009, 5, 502-507;Chen, S et al. ChemBioChem., 2012, 13, 1032-1038; Sako, Y et al. J Am.Chem. Soc., 2008, 130, 7232-7234; Timmerman, P et al. J. Biol. Chem.,2009, 284, 34126-34134).

Described herein are compounds, compositions, and methods useful forsuch purposes.

SUMMARY

Disclosed herein are compounds, compositions, methods for making andscreening large combinatorial libraries of bicyclic peptides displayedon small-molecule scaffolds. In some examples, the overall disk-shapedbicyclic molecules are capable of binding to flat protein surfaces suchas the interfaces of protein-protein interactions. Screening of abicyclic peptide library against tumor necrosis factor-alpha identifiedpotent antagonists that protect cells from tumor necrosisfactor-alpha-induced cell death. Potent K-Ras ligands were alsoidentified from bicyclic libraries.

Disclosed herein are bicyclic peptide compounds. In some examples, thecompounds disclosed herein are anticancer compounds. In some examples,the compounds have been prepared by solid-phase synthesis. In someexamples, the compounds can have a molecular weight of 500 to 5000, suchas from 500 to 2000 or 1000 to 2000.

In some examples, the compounds are of Formula I:

wherein

A is selected from N and benzene;

p, q, and r are independently selected from 0, 1, and 2;

B₁, B₂, and B₃ are independently selected from O and NR¹;

-   -   wherein R¹ comprises H, or substituted or unsubstituted C₁-C₅        alkyl;

L₁ and L₂ are independently selected from hydrogen, substituted orunsubstituted C₁-C₆ alkyl, amino acid, and a linker to a solid phasesupport;

D is selected from hydrogen, substituted or unsubstituted C₁-C₆ alkyl,or amino acid residue; and

X_(m) and X_(n) independently comprise a sequence of 1-10 amino acids.

In some examples of Formula I, the compounds are of Formula I-A:

wherein X_(m) and X_(n) are as defined in Formula I; R⁰ is selected fromhydrogen, substituted or unsubstituted C₁-C₆ alkyl, or linker to solidphase support; and K has a structure represented by a formula:

In some examples of Formula I-A, the compound has a structurerepresented by Formula I-A-1.

In some examples of Formula I-A, the compound has a structurerepresented by Formula I-A-2.

In some examples of Formula I, A comprises N. In some examples ofFormula I, p, q, and r are each 1.

In some examples of Formula I, the compounds are of Formula I-B:

wherein X_(m), X_(n), L¹, L², B¹, B², B³ and D are as defined in FormulaI.

Also disclosed herein, are compounds comprising a) a residue of trimesicacid, the residue bearing three carboxyl functionalities; b) a lysineresidue covalently bonded to the first carboxyl functionality of thetrimesic acid residue; c) a first peptide chain of 1-10 amino acidresidues, X_(m), covalently bonded to the second carboxyl functionalityof the trimesic acid residue; and d) to the lysine residue; and a secondpeptide chain of 1-10 amino acid residues, X_(n), covalently bonded tothe third carboxyl functionality of the trimesic acid residue and to thelysine residue.

Also disclosed is a library comprising a plurality of the disclosedcompounds.

Also disclosed herein is a method for making a bicyclic peptidecompound, the method comprising the steps of: a) linking2,3-diaminopropanoic acid to a solid phase support via its carboxylfunctionality; b) building a first peptide chain of 1-10 amino acidresidues from the 2-amino functionality of the 2,3-diaminopropanoic acidresidue; c) linking a lysine residue to the distal end of the firstpeptide chain; d) building a second peptide chain of 1-10 amino acidresidues onto the lysine residue; e) linking trimesic acid to the3-amino functionality of the 2,3-diaminopropanoic acid residue; f)cyclizing the distal amino acid residue of the second peptide chain witha carboxyl functionality of the trimesic acid; and g) cyclizing theamino side chain of the lysine residue with a carboxyl functionality ofthe trimesic acid.

Also disclosed herein is a method for making a library of bicyclicpeptide compounds, the method comprising the steps of: a) linking2,3-diaminopropanoic acid to a solid phase support via its carboxylfunctionality; b) building a first peptide chain of 1-10 amino acidresidues from the 2-amino functionality of the 2,3-diaminopropanoic acidresidue, using a split-and-pool technique to prepare the chain; c)linking a lysine residue to the distal end of the first peptide chain;d) building a second peptide chain of 1-10 amino acid residues onto thelysine residue, using a split-and-pool technique to prepare the chain;e) linking trimesic acid to the 3-amino functionality of the2,3-diaminopropanoic acid residue; f) cyclizing the distal amino acidresidue of the second peptide chain with a carboxyl functionality of thetrimesic acid; and g) cyclizing the amino side chain of the lysineresidue with a carboxyl functionality of the trimesic acid.

Also disclosed herein is a method of treating or preventing a disorderin a subject, such as a human, comprising administering to the subjectan effective amount of a compound disclosed herein or a pharmaceuticallyacceptable salt thereof. In some examples, the subject is an animal,such as a human. In some examples, the subject is identified as having aneed for treatment of the disorder. In some examples, the method treatsa disorder. In some examples, the disorder is associated withTNF-α-induced cell death, such as dysfunctional regulation ofTNF-α-induced cell death. In some examples the disorder is associatedwith uncontrolled cellular proliferation, such as cancer. In someexamples, the disorder is cancer. In some examples the disorder is aninflammatory disorder. In some examples, the disorder is an autoimmunedisorder, such as a disorder selected from rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitissuppurativa, and refractory asthma.

Also disclosed herein is a method for identifying a drug candidate fortreatment of a disorder, the method comprising the steps of: exposing acompound disclosed herein, a compound prepared by the methods disclosedherein, a library disclosed herein, or a library prepared by the methodsdisclosed to a receptor associated with the disorder; b) detectingreaction between the receptor and the compound or the library; and c)determining the identity of compound reacting with the receptor.

While aspects of the present invention can be described and claimed in aparticular statutory class, such as the system statutory class, this isfor convenience only and one of skill in the art will understand thateach aspect of the present invention can be described and claimed in anystatutory class. Unless otherwise expressly stated, it is in no wayintended that any method or aspect set forth herein be construed asrequiring that its steps be performed in a specific order. Accordingly,where a method claim does not specifically state in the claims ordescriptions that the steps are to be limited to a specific order, it isno way intended that an order be inferred, in any respect. This holdsfor any possible non-express basis for interpretation, including mattersof logic with respect to arrangement of steps or operational flow, plainmeaning derived from grammatical organization or punctuation, or thenumber or type of aspects described in the specification.

DESCRIPTION OF FIGURES

The accompanying figures, which are incorporated in and constitute apart of this specification, illustrate several aspects and together withthe description serve to explain the principles of the invention.

FIG. 1 shows the synthesis of some of the disclosed compounds, such as abicyclic peptide library. Reagents and Conditions: (a) StandardFmoc/HATU chemistry; (b) soak in water; (c) 0.4 equiv Fmoc-OSu inEt₂O/CH₂Cl₂; (d) di-t-butyl dicarbonate; (e) piperidine; (f)4-hydroxybenzoic acid/HBTU/HOBT; (g) Fmoc-13-Ala-OH/DIC; (h) 50% TFA inDCM; (i) split-and-pool synthesis by Fmoc/HATU chemistry; (j) 2% TFA inDCM (6×); (k) Fmoc-OSu/DIPEA in DCM; (l) Pd(PPh₃)₄; (m) diallylprotected trimesic acid/HATU; (n) PyBOP/HOBT/DIPEA; (o) modified reagentK.

FIGS. 2A and 2B show solution-phase screening of initial hits (4^(th)round of screening). FIG. 2A shows selective labeling of bicyclicpeptides with TMR and their release from individual beads by basehydrolysis. FIG. 2B shows evaluation of the 44 released bicyclicpeptides for binding to TNFα in solution by fluorescent anisotropy usinga fixed concentration of TNFα (5 μM) and TMR-labeled bicyclic peptide(100 nM).

FIGS. 3A through 3C show FA analysis of the binding affinity ofresynthesized Necrostatin C1 and C2 for TNFα. FIG. 3A shows thestructures of Necrostatin C1 and C2. FIGS. 3B and 3C are plots of FAvalues as a function of TNFα concentration for Necrostatin C1 and C2,respectively.

FIG. 4 shows Inhibition of TNFα-TNFR1 interaction by Necrostatin C1. Theabsorbance values on the y axis, which reflect the amount of TNFR1 boundto immobilized TNFα in the presence of increasing concentrations ofNecrostatin C1, are relative to that in the absence of peptide inhibitor(100%).

FIG. 5 shows the protection against TNFα-induced cell death byNecrostatin C1. WEHI13VAR cells were incubated overnight at 37° C. inthe presence of TNFα (0.04 ng/mL) and varying concentrations of peptide(0-25 μM), and the number of live cells was quantitated by MTT assay.The absorbance values on the y axis, which reflect the number of livecells, are relative to that of DMSO control (no TNFα, no peptide).

FIG. 6 shows mass spectra showing the quality of the bicyclic peptidelibrary. Ten beads were randomly selected from the library and treatedwith CNBr, and the peptides released were analyzed by MALDI-TOF MS. Eachbead produced a pair of peaks separated by 528 amu.

FIG. 7 shows FA analysis of TNFα Binding by bicyclic peptides releasedfrom single beads. For each bead, the released TMR-labeled bicyclicpeptide (˜50 nM) was incubated with varying concentrations of TNFα (0-18μM) and the FA values are plotted against TNFα concentration. Curvefitting (as described in main text) gave the K_(D) values.

FIGS. 8A and 8B show sequence determination of positive hits by PED-MSfor Necrostatin C1 (FIG. 8A, wherePhg-Tyr-D-Ala-Lys-Tyr-D-Phe-Gly-D-Lys-His is SEQ ID NO: 99) andNectostatin C2 (FIG. 8B, where Ala-D-Phe-Trp-D-Glu-Lys-Nle-D-Leu-Ala-Hisis SEQ ID NO: 100). Positive beads after the solution-phase screening(4^(th) round) were subjected to 11 cycles of PED, and the peptides werereleased from each bead by CNBr, and analyzed by MALDI-TOF MS. M*,homoserine lactone.

FIG. 9A shows the structures of FITC-labeled Necrostatin C1 and C2, thelinear and monocyclic analogs of C1, and a control bicyclic peptide.FIG. 9B shows binding of C1 analogs and control bicyclic peptide to TNFαas determined by FA.

FIGS. 10A and 10B show FA analysis of binding of FITC-labeledNecrostatin C1 (FIG. 10A) and Necrostatin C2 (FIG. 10B) to controlproteins. BSA, bovine serum albumin; BRCT, GST fusion with the BRCTdomain of TopBP1; CA, HIV-1 capsid protein; PLCy2N, GST fusion with theN-SH2 domain of SH2 of PLCγ; and PTP1B, protein tyrosine phosphatase 1B.

FIG. 11 shows competition between Necrostatin C1 and C2 for binding toTNFα. TNFα (1600 nM), FITC-labeled Necrostatin C1 or C2 (100 nM), andvarying concentrations of unlabeled Necrostatin C2 (0-16 μM) wereincubated for 1 h at 37° C. and the FA values were measured and plottedas a function of Necrostatin C2 concentration.

FIG. 12 shows protection of TNFα-induced cell death by Necrostatin C1.WEHI13VAR cells were treated with increasing concentrations of TNFα(0-250 ng/mL) in the absence and presence of 50 μM Necrostatin C1. Afterincubation at 37° C. overnight, the number of live cells was determinedby the MTT assay and plotted as a function of TNFα concentration. All yaxis values are relative to that in the absence of TNFα or NecrostatinC1 (100%).

FIG. 13 displays a schematic of the synthesis of peptide library I.Reagents and Conditions: (a) Standard Fmoc/HBTU chemistry; (b) Soak inwater; (c) 0.5 eq. of Fmoc-OSu; (d) Boc anhydride, DMF; (e) 20%piperidine; (f) Fmoc-Dap(Alloc)-OH, HATU; (g) 95:5 TFA/H₂O; (h) 20%piperidine; (i) Split into 2 equal portions, then standard Fmoc/HATUchemistry; CPP=cell penetrating peptide (j) Pooled the beads, thenFmoc-Lys(Mtt)-OH, HATU; (k) Split and pool synthesis.

FIG. 14 displays the unnatural building blocks used in the library.

FIG. 15 displays a schematic of the preparation of the bicyclic peptidelibrary for screening. Reagents and Conditions: (a) 2% TFA in DCM; (b)Fmoc-OSu; (c) Pd(PPh₃)₄; (d) Click Chemistry: DCAI-N₃, Cu(I); (e)Trimesic acid, HATU; (f) 2% DBU in DMF; (g) pyBOP, HOBt.

FIG. 16 displays a schematic of the synthesis of DCAI-N₃.

FIG. 17 displays the results of a competition assay between FAM-labeledpeptide 2 and DCAI.

FIG. 18 displays the anti-proliferative properties of peptide 41 againstH1299 cells measured by 24-hour MTT assays.

FIG. 19 displays the anti-proliferative properties of peptide 41 againstH1299 cells measured by 72-hour MTT assays.

FIG. 20 shows sequences of hit peptides derived from screening thebicyclic library against K-Ras.

FIG. 21 shows sequences of hit peptides from screening results oflibrary II against biotinylated K-Ras.

FIG. 22a displays structures of cyclorasin B2 and B3. FIG. 22b displaysa FA analysis of K-Ras (mixture of Ras-GTP and Ras-GDP) binding byFITC-labeled cyclorasin B2 and its monocyclic and linear counterparts.FIG. 22c displays a FA analysis of K-Ras binding by cyclorasin B3. FIG.22d is a comparison of FITC-labeled cyclorasin B2 binding to Ras-GTP,Ras-GDP, and Ras-GPPNP. FIG. 22e is a comparison of FITC-labeledcyclorasin B3 binding to Ras-GTP, Ras-GDP, and Ras-GPPNP.

FIGS. 23a through 23f display the biological characterization ofcyclorasin B3. FIGS. 23a and 23b show on-bead assay of inhibition ofRas-Raf interaction by cyclorasin B3. In the absence of inhibitor (FIG.23a ), binding of Texas red-labeled K-Ras (500 nM) to immobilizedGST-Raf RBD rendered the beads red, whereas the addition of 10 μMcyclorasin B3 abolished Ras-Raf interaction (FIG. 23b ). FIG. 23cdisplays the determination of cyclorasin B3 potency by HTRF assay. FIG.23d displays the effect of GST-Raf RBD on FITC-cyclorasin B3 (100 nM)binding to K-Ras (3 μM). FIG. 23e displays the effect of compound 12 onFITC-cyclorasin B3 (100 nM) binding to K-Ras (3 μM). FIG. 23f shows theeffect of oleiylated cyclorasin B3 on the growth rate of H358 lungcancer cells as measured by MTT assay.

Additional advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or can be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION

The present invention can be understood more readily by reference to thefollowing detailed description of the invention and the Examplesincluded therein.

Before the present compounds, compositions, articles, systems, devices,and/or methods are disclosed and described, it is to be understood thatthey are not limited to specific synthetic methods unless otherwisespecified, or to particular reagents unless otherwise specified, as suchmay, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only andis not intended to be limiting. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, example methods andmaterials are now described.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. The publications discussed herein areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing herein is to be construed as an admissionthat the present invention is not entitled to antedate such publicationby virtue of prior invention. Further, the dates of publication providedherein can be different from the actual publication dates, which canrequire independent confirmation.

General Definitions

As used herein, nomenclature for compounds, including organic compounds,can be given using common names, IUPAC, IUBMB, or CAS recommendationsfor nomenclature. When one or more stereochemical features are present,Cahn-Ingold-Prelog rules for stereochemistry can be employed todesignate stereochemical priority, E/Z specification, and the like. Oneof skill in the art can readily ascertain the structure of a compound ifgiven a name, either by systemic reduction of the compound structureusing naming conventions, or by commercially available software, such asCHEMDRAW™ (Cambridgesoft Corporation, U.S.A.).

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a functionalgroup,” “an alkyl,” or “a residue” includes mixtures of two or more suchfunctional groups, alkyls, or residues, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, a further aspect includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms a further aspect. It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that each unit between two particularunits are also disclosed. For example, if 10 and 15 are disclosed, then11, 12, 13, and 14 are also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition or article for which a part by weight isexpressed. Thus, in a compound containing 2 parts by weight of componentX and 5 parts by weight component Y, X and Y are present at a weightratio of 2:5, and are present in such ratio regardless of whetheradditional components are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

As used herein, the terms “optional” or “optionally” means that thesubsequently described event or circumstance can or cannot occur, andthat the description includes instances where said event or circumstanceoccurs and instances where it does not.

As used herein, the term “subject” refers to the target ofadministration, e.g. a subject. Thus the subject of the herein disclosedmethods can be a vertebrate, such as a mammal, a fish, a bird, areptile, or an amphibian. Alternatively, the subject of the hereindisclosed methods can be a human, non-human primate, horse, pig, rabbit,dog, sheep, goat, cow, cat, guinea pig, fish, bird, or rodent. The termdoes not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered. In some examples, the subject is a mammal. A patient refers toa subject afflicted with a disease or disorder. The term “patient”includes human and veterinary subjects. In some examples of thedisclosed methods, the subject has been diagnosed with a need fortreatment of cancer prior to the administering step. In some examples ofthe disclosed method, the subject has been diagnosed with cancer priorto the administering step. The term subject also includes a cell, suchas an animal, for example human, cell.

As used herein, the term “treatment” refers to the medical management ofa patient with the intent to cure, ameliorate, stabilize, or prevent adisease, pathological condition, or disorder. This term includes activetreatment, that is, treatment directed specifically toward theimprovement of a disease, pathological condition, or disorder, and alsoincludes causal treatment, that is, treatment directed toward removal ofthe cause of the associated disease, pathological condition, ordisorder. In addition, this term includes palliative treatment, that is,treatment designed for the relief of symptoms rather than the curing ofthe disease, pathological condition, or disorder; preventativetreatment, that is, treatment directed to minimizing or partially orcompletely inhibiting the development of the associated disease,pathological condition, or disorder; and supportive treatment, that is,treatment employed to supplement another specific therapy directedtoward the improvement of the associated disease, pathologicalcondition, or disorder. In some examples, the term covers any treatmentof a subject, including a mammal (e.g., a human), and includes: (i)preventing the disease from occurring in a subject that can bepredisposed to the disease but has not yet been diagnosed as having it;(ii) inhibiting the disease, i.e., arresting its development; or (iii)relieving the disease, i.e., causing regression of the disease. In someexamples, the subject is a mammal such as a primate, and, in someexamples, the subject is a human. The term “subject” also includesdomesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle,horses, pigs, sheep, goats, fish, bird, etc.), and laboratory animals(e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).

As used herein, the term “prevent” or “preventing” refers to precluding,averting, obviating, forestalling, stopping, or hindering something fromhappening, especially by advance action. It is understood that wherereduce, inhibit or prevent are used herein, unless specificallyindicated otherwise, the use of the other two words is also expresslydisclosed.

As used herein, the term “diagnosed” means having been subjected to aphysical examination by a person of skill, for example, a physician, andfound to have a condition that can be diagnosed or treated by thecompounds, compositions, or methods disclosed herein. For example,“diagnosed with cancer” means having been subjected to a physicalexamination by a person of skill, for example, a physician, and found tohave a condition that can be diagnosed or treated by a compound orcomposition that can treat or prevent cancer. As a further example,“diagnosed with a need for treating or preventing cancer” refers tohaving been subjected to a physical examination by a person of skill,for example, a physician, and found to have a condition characterized bycancer or other disease wherein treating or preventing cancer would bebeneficial to the subject.

As used herein, the phrase “identified to be in need of treatment for adisorder,” or the like, refers to selection of a subject based upon needfor treatment of the disorder. For example, a subject can be identifiedas having a need for treatment of a disorder (e.g., a disorder relatedto cancer) based upon an earlier diagnosis by a person of skill andthereafter subjected to treatment for the disorder. It is contemplatedthat the identification can, In some examples, be performed by a persondifferent from the person making the diagnosis. It is also contemplated,in some examples, that the administration can be performed by one whosubsequently performed the administration.

As used herein, the terms “administering” and “administration” refer toany method of providing a pharmaceutical preparation to a subject. Suchmethods are well known to those skilled in the art and include, but arenot limited to, oral administration, transdermal administration,administration by inhalation, nasal administration, topicaladministration, intravaginal administration, ophthalmic administration,intraaural administration, intracerebral administration, rectaladministration, sublingual administration, buccal administration, andparenteral administration, including injectable such as intravenousadministration, intra-arterial administration, intramuscularadministration, and subcutaneous administration. Administration can becontinuous or intermittent. In some examples, a preparation can beadministered therapeutically; that is, administered to treat an existingdisease or condition. In some examples, a preparation can beadministered prophylactically; that is, administered for prevention of adisease or condition.

The term “contacting” as used herein refers to bringing a disclosedcompound and a cell, target receptor, or other biological entitytogether in such a manner that the compound can affect the activity ofthe target (e.g., receptor, transcription factor, cell, etc.), eitherdirectly; i.e., by interacting with the target itself, or indirectly;i.e., by interacting with another molecule, cofactor, factor, or proteinon which the activity of the target is dependent.

As used herein, the terms “effective amount” and “amount effective”refer to an amount that is sufficient to achieve the desired result orto have an effect on an undesired condition. For example, a“therapeutically effective amount” refers to an amount that issufficient to achieve the desired therapeutic result or to have aneffect on undesired symptoms, but is generally insufficient to causeadverse side effects. The specific therapeutically effective dose levelfor any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the specific composition employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the route ofadministration; the rate of excretion of the specific compound employed;the duration of the treatment; drugs used in combination or coincidentalwith the specific compound employed and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of a compound at levels lower than those required to achievethe desired therapeutic effect and to gradually increase the dosageuntil the desired effect is achieved. If desired, the effective dailydose can be divided into multiple doses for purposes of administration.Consequently, single dose compositions can contain such amounts orsubmultiples thereof to make up the daily dose. The dosage can beadjusted by the individual physician in the event of anycontraindications. Dosage can vary, and can be administered in one ormore dose administrations daily, for one or several days. Guidance canbe found in the literature for appropriate dosages for given classes ofpharmaceutical products. In some examples, a preparation can beadministered in a “prophylactically effective amount”; that is, anamount effective for prevention of a disease or condition.

As used herein, “EC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% enhancement or activation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. EC₅₀ also refers to the concentration or dose of a substance thatis required for 50% enhancement or activation in vivo, as furtherdefined elsewhere herein. Alternatively, EC₅₀ can refer to theconcentration or dose of compound that provokes a response halfwaybetween the baseline and maximum response. The response can be measuredin an in vitro or in vivo system as is convenient and appropriate forthe biological response of interest. For example, the response can bemeasured in vitro using cultured muscle cells or in an ex vivo organculture system with isolated muscle fibers. Alternatively, the responsecan be measured in vivo using an appropriate research model such asrodent, including mice and rats. The mouse or rat can be an inbredstrain with phenotypic characteristics of interest such as obesity ordiabetes. As appropriate, the response can be measured in a transgenicor knockout mouse or rat wherein the gene or genes has been introducedor knocked-out, as appropriate, to replicate a disease process.

As used herein, “IC₅₀,” is intended to refer to the concentration ordose of a substance (e.g., a compound or a drug) that is required for50% inhibition or diminuation of a biological process, or component of aprocess, including a protein, subunit, organelle, ribonucleoprotein,etc. IC₅₀ also refers to the concentration or dose of a substance thatis required for 50% inhibition or diminuation in vivo, as furtherdefined elsewhere herein. Alternatively, IC₅₀ also refers to the halfmaximal (50%) inhibitory concentration (IC) or inhibitory dose of asubstance. The response can be measured in an in vitro or in vivo systemas is convenient and appropriate for the biological response ofinterest. For example, the response can be measured in vitro usingcultured muscle cells or in an ex vivo organ culture system withisolated muscle fibers. Alternatively, the response can be measured invivo using an appropriate research model such as rodent, including miceand rats. The mouse or rat can be an inbred strain with phenotypiccharacteristics of interest such as obesity or diabetes. As appropriate,the response can be measured in a transgenic or knockout mouse or ratwherein a gene or genes has been introduced or knocked-out, asappropriate, to replicate a disease process.

The term “pharmaceutically acceptable” describes a material that is notbiologically or otherwise undesirable, i.e., without causing anunacceptable level of undesirable biological effects or interacting in adeleterious manner.

As used herein, the term “derivative” refers to a compound having astructure derived from the structure of a parent compound (e.g., acompound disclosed herein) and whose structure is sufficiently similarto those disclosed herein and based upon that similarity, would beexpected by one skilled in the art to exhibit the same or similaractivities and utilities as the claimed compounds, or to induce, as aprecursor, the same or similar activities and utilities as the claimedcompounds. Exemplary derivatives include salts, esters, amides, salts ofesters or amides, and N-oxides of a parent compound.

As used herein, the term “pharmaceutically acceptable carrier” refers tosterile aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, as well as sterile powders for reconstitution into sterileinjectable solutions or dispersions just prior to use. Examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol and the like), carboxymethylcellulose and suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of coating materials such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants. These compositions can also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of the action of microorganisms can be ensured by theinclusion of various antibacterial and antifungal agents such asparaben, chlorobutanol, phenol, sorbic acid and the like. It can also bedesirable to include isotonic agents such as sugars, sodium chloride andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the inclusion of agents, such as aluminummonostearate and gelatin, which delay absorption. Injectable depot formsare made by forming microencapsule matrices of the drug in biodegradablepolymers such as polylactide-polyglycolide, poly(orthoesters) andpoly(anhydrides). Depending upon the ratio of drug to polymer and thenature of the particular polymer employed, the rate of drug release canbe controlled. Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissues. The injectable formulations can be sterilized, forexample, by filtration through a bacterial-retaining filter or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable media just prior to use. Suitable inertcarriers can include sugars such as lactose. Desirably, at least 95% byweight of the particles of the active ingredient have an effectiveparticle size in the range of 0.01 to 10 micrometers.

A residue of a chemical species, as used in the specification andconcluding claims, refers to the moiety that is the resulting product ofthe chemical species in a particular reaction scheme or subsequentformulation or chemical product, regardless of whether the moiety isactually obtained from the chemical species. Thus, an ethylene glycolresidue in a polyester refers to one or more —OCH₂CH₂O— units in thepolyester, regardless of whether ethylene glycol was used to prepare thepolyester. Similarly, a sebacic acid residue in a polyester refers toone or more —CO(CH₂)₈CO— moieties in the polyester, regardless ofwhether the residue is obtained by reacting sebacic acid or an esterthereof to obtain the polyester.

Chemical Definitions

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In some examples, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. It is also contemplated that, in certain examples,unless expressly indicated to the contrary, individual substituents canbe further optionally substituted (i.e., further substituted orunsubstituted).

In defining various terms, “A¹,” “A²,” “A³,” and “A⁴” are used herein asgeneric symbols to represent various specific substituents. Thesesymbols can be any substituent, not limited to those disclosed herein,and when they are defined to be certain substituents in one instance,they can, in another instance, be defined as some other substituents.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl,isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl,dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. Thealkyl group can be cyclic or acyclic. The alkyl group can be branched orunbranched. The alkyl group can also be substituted or unsubstituted.For example, the alkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, cycloalkyl, alkoxy, amino, ether,halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol, as described herein.A “lower alkyl” group is an alkyl group containing from one to six(e.g., from one to four) carbon atoms.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” or “haloalkyl” specifically refers to analkyl group that is substituted with one or more halide, e.g., fluorine,chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refersto an alkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, norbornyl, and the like. The term “heterocycloalkyl” is atype of cycloalkyl group as defined above, and is included within themeaning of the term “cycloalkyl,” where at least one of the carbon atomsof the ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group andheterocycloalkyl group can be substituted or unsubstituted. Thecycloalkyl group and heterocycloalkyl group can be substituted with oneor more groups including, but not limited to, alkyl, cycloalkyl, alkoxy,amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol asdescribed herein.

The term “polyalkylene group” as used herein is a group having two ormore CH₂ groups linked to one another. The polyalkylene group can berepresented by the formula —(CH₂)_(a)—, where “a” is an integer of from2 to 500.

The terms “alkoxy” and “alkoxyl” as used herein to refer to an alkyl orcycloalkyl group bonded through an ether linkage; that is, an “alkoxy”group can be defined as —OA¹ where A¹ is alkyl or cycloalkyl as definedabove. “Alkoxy” also includes polymers of alkoxy groups as justdescribed; that is, an alkoxy can be a polyether such as —OA¹-OA² or—OA¹-(OA²)_(a)-OA³, where “a” is an integer of from 1 to 200 and A¹, A²,and A³ are alkyl and/or cycloalkyl groups.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (A¹A²)C═C(A³A⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, orthiol, as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onecarbon-carbon double bound, i.e., C═C. Examples of cycloalkenyl groupsinclude, but are not limited to, cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl,norbornenyl, and the like. The term “heterocycloalkenyl” is a type ofcycloalkenyl group as defined above, and is included within the meaningof the term “cycloalkenyl,” where at least one of the carbon atoms ofthe ring is replaced with a heteroatom such as, but not limited to,nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group andheterocycloalkenyl group can be substituted or unsubstituted. Thecycloalkenyl group and heterocycloalkenyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be unsubstituted orsubstituted with one or more groups including, but not limited to,alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether,halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiol, asdescribed herein.

The term “cycloalkynyl” as used herein is a non-aromatic carbon-basedring composed of at least seven carbon atoms and containing at least onecarbon-carbon triple bound. Examples of cycloalkynyl groups include, butare not limited to, cycloheptynyl, cyclooctynyl, cyclononynyl, and thelike. The term “heterocycloalkynyl” is a type of cycloalkenyl group asdefined above, and is included within the meaning of the term“cycloalkynyl,” where at least one of the carbon atoms of the ring isreplaced with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkynyl group andheterocycloalkynyl group can be substituted or unsubstituted. Thecycloalkynyl group and heterocycloalkynyl group can be substituted withone or more groups including, but not limited to, alkyl, cycloalkyl,alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,azide, nitro, silyl, sulfo-oxo, or thiol as described herein.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” alsoincludes “heteroaryl,” which is defined as a group that contains anaromatic group that has at least one heteroatom incorporated within thering of the aromatic group. Examples of heteroatoms include, but are notlimited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term“non-heteroaryl,” which is also included in the term “aryl,” defines agroup that contains an aromatic group that does not contain aheteroatom. The aryl group can be substituted or unsubstituted. The arylgroup can be substituted with one or more groups including, but notlimited to, alkyl, cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester,ether, halide, hydroxy, ketone, azide, nitro, silyl, sulfo-oxo, or thiolas described herein. The term “biaryl” is a specific type of aryl groupand is included in the definition of “aryl.” Biaryl refers to two arylgroups that are bound together via a fused ring structure, as innaphthalene, or are attached via one or more carbon-carbon bonds, as inbiphenyl.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” is a short hand notation for acarbonyl group, i.e., C═O.

The terms “amine” or “amino” as used herein are represented by theformula —NA¹A², where A¹ and A² can be, independently, hydrogen oralkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “alkylamino” as used herein is represented by the formula—NH(-alkyl) where alkyl is a described herein. Representative examplesinclude, but are not limited to, methylamino group, ethylamino group,propylamino group, isopropylamino group, butylamino group, isobutylaminogroup, (sec-butyl)amino group, (tert-butyl)amino group, pentylaminogroup, isopentylamino group, (tert-pentyl)amino group, hexylamino group,and the like.

The term “dialkylamino” as used herein is represented by the formula—N(-alkyl)₂ where alkyl is a described herein. Representative examplesinclude, but are not limited to, dimethylamino group, diethylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di(sec-butyl)amino group, di(tert-butyl)aminogroup, dipentylamino group, diisopentylamino group, di(tert-pentyl)aminogroup, dihexylamino group, N-ethyl-N-methylamino group,N-methyl-N-propylamino group, N-ethyl-N-propylamino group and the like.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH.

The term “ester” as used herein is represented by the formula —OC(O)A¹or —C(O)OA¹, where A¹ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein.The term “polyester” as used herein is represented by the formula-(A¹O(O)C-A²-C(O)O)_(a)— or -(A¹O(O)C-A²-OC(O))_(a)—, where A¹ and A²can be, independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl,alkynyl, cycloalkynyl, aryl, or heteroaryl group described herein and“a” is an integer from 1 to 500. “Polyester” is as the term used todescribe a group that is produced by the reaction between a compoundhaving at least two carboxylic acid groups with a compound having atleast two hydroxyl groups.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group describedherein. The term “polyether” as used herein is represented by theformula -(A¹O-A²O)_(n)—, where A¹ and A² can be, independently, analkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group described herein and “a” is an integer of from 1 to500. Examples of polyether groups include polyethylene oxide,polypropylene oxide, and polybutylene oxide.

The term “halide” as used herein refers to the halogens fluorine,chlorine, bromine, and iodine.

The term “heterocycle,” as used herein refers to single and multi-cyclicaromatic or non-aromatic ring systems in which at least one of the ringmembers is other than carbon. Heterocycle includes azetidine, dioxane,furan, imidazole, isothiazole, isoxazole, morpholine, oxazole, oxazole,including, 1,2,3-oxadiazole, 1,2,5-oxadiazole and 1,3,4-oxadiazole,piperazine, piperidine, pyrazine, pyrazole, pyridazine, pyridine,pyrimidine, pyrrole, pyrrolidine, tetrahydrofuran, tetrahydropyran,tetrazine, including 1,2,4,5-tetrazine, tetrazole, including1,2,3,4-tetrazole and 1,2,4,5-tetrazole, thiadiazole, including,1,2,3-thiadiazole, 1,2,5-thiadiazole, and 1,3,4-thiadiazole, thiazole,thiophene, triazine, including 1,3,5-triazine and 1,2,4-triazine,triazole, including, 1,2,3-triazole, 1,3,4-triazole, and the like.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “ketone” as used herein is represented by the formula A¹C(O)A²,where A¹ and A² can be, independently, an alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group asdescribed herein.

The term “azide” as used herein is represented by the formula —N₃.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “nitrile” as used herein is represented by the formula —CN.

The term “silyl” as used herein is represented by the formula —SiA¹A²A³,where A¹, A², and A³ can be, independently, hydrogen or an alkyl,cycloalkyl, alkoxy, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl,or heteroaryl group as described herein.

The term “sulfo-oxo” as used herein is represented by the formulas—S(O)A¹, —S(O)₂A¹, —OS(O)₂A¹, or -0S(O)₂OA¹, where A¹ can be hydrogen oran alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl,aryl, or heteroaryl group as described herein. Throughout thisspecification “S(O)” is a short hand notation for S═O. The term“sulfonyl” is used herein to refer to the sulfo-oxo group represented bythe formula —S(O)₂A¹, where A¹ can be hydrogen or an alkyl, cycloalkyl,alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl groupas described herein. The term “sulfone” as used herein is represented bythe formula A¹S(O)₂A², where A¹ and A² can be, independently, an alkyl,cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, orheteroaryl group as described herein. The term “sulfoxide” as usedherein is represented by the formula A¹S(O)A², where A¹ and A² can be,independently, an alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl,cycloalkynyl, aryl, or heteroaryl group as described herein.

The term “thiol” as used herein is represented by the formula —SH.

“R¹,” “R²,” “R³,” “R^(n),” where n is an integer, as used herein can,independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an alkyl group, a halide, and the like.Depending upon the groups that are selected, a first group can beincorporated within second group or, alternatively, the first group canbe pendant (i.e., attached) to the second group. For example, with thephrase “an alkyl group comprising an amino group,” the amino group canbe incorporated within the backbone of the alkyl group. Alternatively,the amino group can be attached to the backbone of the alkyl group. Thenature of the group(s) that is (are) selected will determine if thefirst group is embedded or attached to the second group.

As described herein, compounds may contain “optionally substituted”moieties. In general, the term “substituted,” whether preceded by theterm “optionally” or not, means that one or more hydrogens of thedesignated moiety are replaced with a suitable substituent. Unlessotherwise indicated, an “optionally substituted” group may have asuitable substituent at each substitutable position of the group, andwhen more than one position in any given structure may be substitutedwith more than one substituent selected from a specified group, thesubstituent may be either the same or different at every position.Combinations of substituents envisioned herein are preferably those thatresult in the formation of stable or chemically feasible compounds. Inis also contemplated that, in some examples, unless expressly indicatedto the contrary, individual substituents can be further optionallysubstituted (i.e., further substituted or unsubstituted).

The term “stable,” as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, and, in some examples, their recovery,purification, and use for one or more of the purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH2)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)⁰⁻⁴C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)₀₋₄R^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight or branchedalkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(●), -(haloR^(●)),—(CH₂)₀₋₂OH, —(CH₂)O₂OR^(●), —(CH₂)₀₋₂CH(OR^(●))₂; —O(haloR^(●)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(●), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(●),—(CH₂)₀₋₂SR^(●), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(●),—(CH₂)₀₋₂NR^(●) ₂, —NO₂, —SiR^(●) ₃, —OSiR^(●) ₃, —C(O)SR, —(C₁₋₄straight or branched alkylene)C(O)OR, or —SSR^(●) wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN, —C(O)OH,—C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein each R^(●) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen, —R^(●), -(haloR^(●)), —OH, —OR^(●), —O(haloR^(●)), —CN,—C(O)OH, —C(O)OR^(●), —NH₂, —NHR^(●), —NR^(●) ₂, or —NO₂, wherein eachR^(●) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

The term “leaving group” refers to an atom (or a group of atoms) withelectron withdrawing ability that can be displaced as a stable species,taking with it the bonding electrons. Examples of suitable leavinggroups include halides and sulfonate esters, including, but not limitedto, triflate, mesylate, tosylate, and brosylate.

The terms “hydrolysable group” and “hydrolysable moiety” refer to afunctional group capable of undergoing hydrolysis, e.g., under basic oracidic conditions. Examples of hydrolysable residues include, withoutlimitation, acid halides, activated carboxylic acids, and variousprotecting groups known in the art (see, for example, “Protective Groupsin Organic Synthesis,” T. W. Greene, P. G. M. Wuts, Wiley-Interscience,1999).

The term “organic residue” defines a carbon containing residue, i.e., aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic residues can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbonatoms, or 1 to 4 carbon atoms. In some examples, an organic residue cancomprise 2 to 18 carbon atoms, 2 to 15, carbon atoms, 2 to 12 carbonatoms, 2 to 8 carbon atoms, 2 to 4 carbon atoms, or 2 to 4 carbon atoms.

A very close synonym of the term “residue” is the term “radical,” whichas used in the specification and concluding claims, refers to afragment, group, or substructure of a molecule described herein,regardless of how the molecule is prepared. For example, a2,4-thiazolidinedione radical in a particular compound has the structure

regardless of whether thiazolidinedione is used to prepare the compound.In some embodiments the radical (for example an alkyl) can be furthermodified (i.e., substituted alkyl) by having bonded thereto one or more“substituent radicals.” The number of atoms in a given radical is notcritical to the compounds and compositions disclosed herein unless it isindicated to the contrary elsewhere herein.

“Organic radicals,” as the term is defined and used herein, contain oneor more carbon atoms. An organic radical can have, for example, 1-26carbon atoms, 1-18 carbon atoms, 1-12 carbon atoms, 1-8 carbon atoms,1-6 carbon atoms, or 1-4 carbon atoms. In some examples, an organicradical can have 2-26 carbon atoms, 2-18 carbon atoms, 2-12 carbonatoms, 2-8 carbon atoms, 2-6 carbon atoms, or 2-4 carbon atoms. Organicradicals often have hydrogen bound to at least some of the carbon atomsof the organic radical. One example, of an organic radical thatcomprises no inorganic atoms is a 5, 6, 7, 8-tetrahydro-2-naphthylradical. In some embodiments, an organic radical can contain 1-10inorganic heteroatoms bound thereto or therein, including halogens,oxygen, sulfur, nitrogen, phosphorus, and the like. Examples of organicradicals include but are not limited to an alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, mono-substituted amino,di-substituted amino, acyloxy, cyano, carboxy, carboalkoxy,alkylcarboxamide, substituted alkylcarboxamide, dialkylcarboxamide,substituted dialkylcarboxamide, alkylsulfonyl, alkylsulfinyl, thioalkyl,thiohaloalkyl, alkoxy, substituted alkoxy, haloalkyl, haloalkoxy, aryl,substituted aryl, heteroaryl, heterocyclic, or substituted heterocyclicradicals, wherein the terms are defined elsewhere herein. A fewnon-limiting examples of organic radicals that include heteroatomsinclude alkoxy radicals, trifluoromethoxy radicals, acetoxy radicals,dimethylamino radicals and the like.

“Inorganic radicals,” as the term is defined and used herein, contain nocarbon atoms and therefore comprise only atoms other than carbon.Inorganic radicals comprise bonded combinations of atoms selected fromhydrogen, nitrogen, oxygen, silicon, phosphorus, sulfur, selenium, andhalogens such as fluorine, chlorine, bromine, and iodine, which can bepresent individually or bonded together in their chemically stablecombinations. Inorganic radicals have 10 or fewer, or preferably one tosix or one to four inorganic atoms as listed above bonded together.Examples of inorganic radicals include, but not limited to, amino,hydroxy, halogens, nitro, thiol, sulfate, phosphate, and like commonlyknown inorganic radicals. The inorganic radicals do not have bondedtherein the metallic elements of the periodic table (such as the alkalimetals, alkaline earth metals, transition metals, lanthanide metals, oractinide metals), although such metal ions can sometimes serve as apharmaceutically acceptable cation for anionic inorganic radicals suchas a sulfate, phosphate, or like anionic inorganic radical. Inorganicradicals do not comprise metalloids elements such as boron, aluminum,gallium, germanium, arsenic, tin, lead, or tellurium, or the noble gaselements, unless otherwise specifically indicated elsewhere herein.

Compounds described herein can contain one or more double bonds and,thus, potentially give rise to cis/trans (E/Z) isomers, as well as otherconformational isomers. Unless stated to the contrary, the compounds andcompositions disclosed herein include all such possible isomers, as wellas mixtures of such isomers.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer and diastereomer, and a mixtureof isomers, such as a racemic or scalemic mixture. Compounds describedherein can contain one or more asymmetric centers and, thus, potentiallygive rise to diastereomers and optical isomers. Unless stated to thecontrary, the compounds and compositions disclosed herein include allsuch possible diastereomers as well as their racemic mixtures, theirsubstantially pure resolved enantiomers, all possible geometric isomers,and pharmaceutically acceptable salts thereof Mixtures of stereoisomers,as well as isolated specific stereoisomers, are also included. Duringthe course of the synthetic procedures used to prepare such compounds,or in using racemization or epimerization procedures known to thoseskilled in the art, the products of such procedures can be a mixture ofstereoisomers.

Many organic compounds exist in optically active forms having theability to rotate the plane of plane-polarized light. In describing anoptically active compound, the prefixes D and L or R and S are used todenote the absolute configuration of the molecule about its chiralcenter(s). The prefixes d and 1 or (+) and (−) are employed to designatethe sign of rotation of plane-polarized light by the compound, with (−)or meaning that the compound is levorotatory. A compound prefixed with(+) or d is dextrorotatory. For a given chemical structure, thesecompounds, called stereoisomers, are identical except that they arenon-superimposable mirror images of one another. A specific stereoisomercan also be referred to as an enantiomer, and a mixture of such isomersis often called an enantiomeric mixture. A 50:50 mixture of enantiomersis referred to as a racemic mixture. Many of the compounds describedherein can have one or more chiral centers and therefore can exist indifferent enantiomeric forms. If desired, a chiral carbon can bedesignated with an asterisk (*). When bonds to the chiral carbon aredepicted as straight lines in the disclosed formulas, it is understoodthat both the (R) and (S) configurations of the chiral carbon, and henceboth enantiomers and mixtures thereof, are embraced within the formula.As is used in the art, when it is desired to specify the absoluteconfiguration about a chiral carbon, one of the bonds to the chiralcarbon can be depicted as a wedge (bonds to atoms above the plane) andthe other can be depicted as a series or wedge of short parallel linesis (bonds to atoms below the plane). The Cahn-Inglod-Prelog system canbe used to assign the (R) or (S) configuration to a chiral carbon.

Compounds described herein comprise atoms in both their natural isotopicabundance and in non-natural abundance. The disclosed compounds can beisotopically-labelled or isotopically-substituted compounds identical tothose described, but for the fact that one or more atoms are replaced byan atom having an atomic mass or mass number different from the atomicmass or mass number typically found in nature. Examples of isotopes thatcan be incorporated into compounds disclosed herein include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine and chlorine,such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³⁵S, ¹⁸F and ³⁶Cl respectively.Compounds further comprise prodrugs thereof, and pharmaceuticallyacceptable salts of said compounds or of said prodrugs which contain theaforementioned isotopes and/or other isotopes of other atoms are withinthe scope of this invention. Certain isotopically-labeled compounds, forexample those into which radioactive isotopes such as ³H and ¹⁴C areincorporated, are useful in drug and/or substrate tissue distributionassays. Tritiated, i.e., ³H, and carbon-14, i.e., ¹⁴C, isotopes areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium, i.e., ²H,can afford certain therapeutic advantages resulting from greatermetabolic stability, for example increased in vivo half-life or reduceddosage requirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds and prodrugs thereof can generally beprepared by carrying out the procedures below, by substituting a readilyavailable isotopically labeled reagent for a non-isotopically labeledreagent.

The compounds described herein can be present as a solvate. In somecases, the solvent used to prepare the solvate is an aqueous solution,and the solvate is then often referred to as a hydrate. The compoundscan be present as a hydrate, which can be obtained, for example, bycrystallization from a solvent or from aqueous solution. In thisconnection, one, two, three or any arbitrary number of solvate or watermolecules can combine with the compounds disclosed herein to formsolvates and hydrates. Unless stated to the contrary, all such possiblesolvates are included in the discussion herein.

The term “co-crystal” means a physical association of two or moremolecules which owe their stability through non-covalent interaction.One or more components of this molecular complex provide a stableframework in the crystalline lattice. In certain instances, the guestmolecules are incorporated in the crystalline lattice as anhydrates orsolvates, see e.g. “Crystal Engineering of the Composition ofPharmaceutical Phases. Do Pharmaceutical Co-crystals Represent a NewPath to Improved Medicines?” Almarasson, O., et. al., The Royal Societyof Chemistry, 1889-1896, 2004. Examples of co-crystals includep-toluenesulfonic acid and benzenesulfonic acid.

It is also appreciated that certain compounds described herein can bepresent as an equilibrium of tautomers. For example, ketones with ana-hydrogen can exist in an equilibrium of the keto form and the enolform.

Likewise, amides with an N-hydrogen can exist in an equilibrium of theamide form and the imidic acid form. Unless stated to the contrary, allsuch possible tautomers are included herein.

It is known that chemical substances form solids which are present indifferent states of order which are termed polymorphic forms ormodifications. The different modifications of a polymorphic substancecan differ greatly in their physical properties. The compounds can bepresent in different polymorphic forms, with it being possible forparticular modifications to be metastable. Unless stated to thecontrary, all such possible polymorphic forms are included.

In some examples, a structure of a compound can be represented by aformula:

which is understood to be equivalent to a formula:

wherein n is typically an integer. That is, R^(n) is understood torepresent five independent substituents, R^(n(a)), R^(n(b)), R^(n(c)),R^(n(d)), R^(n(e)). By “independent substituents,” it is meant that eachR substituent can be independently defined. For example, if in oneinstance R^(n(a)) is halogen, then R^((b)) is not necessarily halogen inthat instance.

Certain materials, compounds, compositions, and components disclosedherein can be obtained commercially or readily synthesized usingtechniques generally known to those of skill in the art. For example,the starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), orSigma (St. Louis, Mo.) or are prepared by methods known to those skilledin the art following procedures set forth in references such as Fieserand Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wileyand Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989).

Unless otherwise expressly stated, it is in no way intended that anymethod set forth herein be construed as requiring that its steps beperformed in a specific order. Accordingly, where a method claim doesnot actually recite an order to be followed by its steps or it is nototherwise specifically stated in the claims or descriptions that thesteps are to be limited to a specific order, it is no way intended thatan order be inferred, in any respect. This holds for any possiblenon-express basis for interpretation, including: matters of logic withrespect to arrangement of steps or operational flow; plain meaningderived from grammatical organization or punctuation; and the number ortype of embodiments described in the specification.

Disclosed are the components to be used to prepare the compositionsdisclosed herein as well as the compositions themselves to be usedwithin the methods disclosed herein. These and other materials aredisclosed herein, and it is understood that when combinations, subsets,interactions, groups, etc. of these materials are disclosed that whilespecific reference of each various individual and collectivecombinations and permutation of these compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular compound is disclosed and discussed and anumber of modifications that can be made to a number of moleculesincluding the compounds are discussed, specifically contemplated is eachand every combination and permutation of the compound and themodifications that are possible unless specifically indicated to thecontrary. Thus, if a class of molecules A, B, and C are disclosed aswell as a class of molecules D, E, and F and an example of a combinationmolecule, A-D is disclosed, then even if each is not individuallyrecited each is individually and collectively contemplated meaningcombinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considereddisclosed. Likewise, any subset or combination of these is alsodisclosed. Thus, for example, the sub-group of A-E, B-F, and C-E wouldbe considered disclosed. This concept applies to all aspects of thisapplication including, but not limited to, steps in methods of makingand using the compositions disclosed herein. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the methods disclosedherein.

It is understood that the compositions disclosed herein have certainfunctions. Disclosed herein are certain structural requirements forperforming the disclosed functions, and it is understood that there area variety of structures that can perform the same function that arerelated to the disclosed structures, and that these structures willtypically achieve the same result.

Tumor Necrosis Factor-Alpha

Tumor necrosis factor-alpha (TNFα) is a pleiotropic inflammatorycytokine of a variety of functions, many of which are not yet fullyunderstood (Chen, G and Goeddel, D V. Science, 2002, 296, 1634-1635).TNFα is responsible for cachexia, wasting in patients with chronicdiseases such as cancer and tuberculosis (Kawakami, M and Cerami, A. J.Exp. Med., 1981, 154, 631-639) and is implicated in the development ofseptic shock and multi-organ failure in severely infected patients(Beutler, B et al. J. Exp. Med., 1985, 161, 984-995). It is alsoresponsible for numerous chronic inflammatory disorders such asrheumatoid arthritis, ankylosing spondylitis, inflammatory boweldisease, psoriasis, hidradenitis suppurativa, and refractory asthma(Esposito, E and Cuzzocrea, S. Curr. Med. Chem., 2009, 16, 3152-3167).These disorders are currently treated with protein inhibitors, includingmonoclonal antibodies infliximab (Remicade), adalimumab (Humira) orcertolizumab pegol (Cimzia), and a circulating receptor fusion proteinetanercept (Enbrel). These proteins bind specifically to TNFα andprevent its interaction with TNFα receptors (TNFRs). These biologicdrugs are administered by in-hospital intravenous injections.Considerable efforts have been made over the past two decades to developsmall-molecule inhibitors against TNFα, which have the potential to beadministered orally. However, these efforts have so far led to only afew weak small-molecule inhibitors that directly bind to TNFα andinterfere with TNFα-TNFR interaction (Alzani, R et al. J. Biol. Chem.,1993, 268, 12526-12529; Mancini, F et al. Biochem. Pharmocol., 1999, 58,851-859; He, M M et al. Science, 2005, 310, 1022-1025; Chan, D S et al.Angew. Chem. Int. Ed. Engi., 2010, 49, 2860-2864; Choi, H et al. Bioorg.Med. Chem. Lett., 2010, 20, 6195-6198; Buller, F et al. Chem. Biol.,2009, 16, 1075-1086; Leung, C H et al. ChemMedChem, 2011, 6, 765-768).Disulfide-mediated cyclic peptides corresponding to the TNFα-bindingsites in TNFR1 have also shown weak inhibitory activity against TNFαbinding to its receptor and TNFα-mediated apoptosis (Takasaki, W et al.Nat. Biotechnol., 1997, 15, 1266-1270; Saito, H et al. Arthritis Rheum.,2007, 56, 1164-1174). The latter observation inspired us to developmacrocyclic compounds as TNFα inhibitors. Disclosed herein is amethodology for high-throughput synthesis and screening of largecombinatorial libraries of bicyclic peptides and the discovery ofNecrostatin C1 as a potent, bicyclic peptidyl antagonist against TNFα.

Disclosed herein is a methodology for chemical synthesis and screeningof large combinatorial libraries of bicyclic peptides displayed onsmall-molecule scaffolds. By using planar trimesic acid as the scaffold,it is shown that the resulting disk-shaped molecules are privileged forbinding to flat protein surfaces such as the interfaces ofprotein-protein interactions. Screening of a bicyclic peptide libraryagainst tumor necrosis factor-alpha (TNFα) identified a potentantagonist that inhibits the TNFα-TNFα receptor interaction and protectscells from TNFα-induced cell death. Bicyclic peptides of this type canprovide a general solution for inhibition of protein-proteininteractions.

In some examples, disclosed herein is a method for displaying peptidicor peptidomimetic sequences on small-molecule scaffolds to form bicyclicmolecules that rival antibodies for binding affinity and specificity.Inhibitors were developed against PPIs, which are a large class ofexciting but generally considered “undruggable” targets by conventionalsmall-molecule approaches. A planar scaffold, trimesic acid, was chosenin order to maximize the surface area of the resulting molecules andtherefore their ability to interact with flat protein surfaces such asthe PPI interfaces. A bicyclic peptide library was generated by“wrapping” a peptide sequence of 6-10 random residues around thetrimesoyl group. Peptide cyclization was mediated by the formation threeamide bonds between the trimesoyl scaffold and the N-terminal amine, theside chain of a C-terminal L-2, 3-diaminopropionic acid (Dap), and theside chain of a fixed lysine within the random region. The resultingbicyclic peptides contained 3-5 random residues in each ring. The randomsequence was constructed with a 25-amino acid set judiciously selectedbased on their structural diversity, metabolic stability, and commercialavailability. It included 10 proteinogenic α-L-amino acids [Ala, Arg,Asp, Gin, Gly, His, Ile, Ser, Trp, and Tyr], 5 nonproteinogenicα-L-amino acids [L-4-fluorophenylalanine (Fpa), L-norleucine (Nle),L-omithine (Orn), and L-phenylglycine (Phg)], and 10 a-D-amino acids[D-2-naphthylalanine (D-Nal), D-Ala, D-Asn, D-Glu, D-Leu, D-Lys, D-Phe,D-Pro, D-Thr, and D-Val]. This library has a theoretical diversity of1.0×10e14. Inclusion of unnatural amino acids (and in the futurenon-peptidic building blocks) greatly increases the structural diversityand proteolytic stability of the compounds but also necessitates thechemical synthesis of the resulting compound libraries.

A challenge associated with screening chemically synthesized bicyclicpeptide libraries is structural determination of the hit compounds. Toovercome this difficulty, the bicyclic peptide library was synthesizedin the one bead-two compound format on TentaGel microbeads (90 μm,2.86×10e6 beads/g, ˜100 pmol peptide/bead), by employing a publishedbead-segregation technique. Each library bead was topologicallysegregated into two different layers, with the outer layer displaying aunique bicyclic peptide and the inner layer containing the correspondinglinear peptide as an encoding tag. The symmetry of the trimesoyl unitensured that a single bicyclic product was formed on each bead. Thelibrary was designed such that the bicyclic peptide on the bead surfacealso carried an L-propargylglycine (Pra) residue in its linker region,whereas the linear encoding peptide does not.

Library screening against target proteins can be carried out in foursteps. During steps 1 and 2, the library was incubated with the targetprotein labeled with a biotin and positive beads were isolated bymagnetic sorting (step 1) and an on-bead enzyme-linked assay (step 2).At step 3, the positive beads from step 2 were tested again againstfluorescently labeled target protein and the fluorescent beads wereisolated. Finally, at step 4 the bicyclic peptide was selectivelyreleased from each positive bead and tested for binding to the targetprotein in solution by fluorescence polarization assay. For bicyclicpeptides that showed solution-phase binding activity, the encodingpeptides on their corresponding beads were individually sequenced by amass spectrometric method previously developed in this laboratory(PED-MS).

Screening of the bicyclic peptide library against human tumor necrosisfactor-alpha (TNF-alpha) resulted in two potent ligands, Necrostatin C1and C2, with K_(D) values of 0.45 and 1.9 μM, respectively. NecrostatinC1 was selected for further tests and shown to inhibit TNF-alpha bindingto its cognate receptor with an IC₅₀ of 3.1 micromolar. It alsoprotected cells from TNF-alpha induced cell death. The same library wasalso screened against human K-Ras protein. Several high nanomolar K-Rasligands were discovered, some of which inhibited the interaction betweenK-Ras and its effector proteins (e.g., Raf kinase). The TNF-alphainhibitors are potentially useful for treatment of inflammatory diseasessuch as rheumatoid arthritis. K-Ras inhibitors will provide a novelclass of anticancer drugs.

Compounds

Disclosed herein are bicyclic peptide compounds. In some examples, thecompounds disclosed herein are anticancer compounds. In some examples,the compounds have been prepared by solid-phase synthesis. In someexamples, the compounds can have a molecular weight of 500 to 5000, suchas from 500 to 2000 or 1000 to 2000.

In some examples, the compounds are of Formula I:

wherein

A is selected from N and benzene;

p, q, and r are independently selected from 0, 1, and 2;

B₁, B₂, and B₃ are independently selected from O and NR¹;

wherein R¹ comprises H, or substituted or unsubstituted C₁-C₅ alkyl;

L₁ and L₂ are independently selected from hydrogen, substituted orunsubstituted C₁-C₆ alkyl, amino acid, and a linker to a solid phasesupport;

D is selected from hydrogen, substituted or unsubstituted C₁-C₆ alkyl,or amino acid residue; and

X_(m) and X_(n) independently comprise a sequence of 1-10 amino acids.

In some examples, the compounds disclosed herein of Formula I cancomprise anticancer compounds. In some examples, the compounds ofFormula I can be prepared by solid-phase synthesis.

In some examples, the compounds of Formula I can have a molecular weightof 500 or greater (e.g., 1000 or greater, 1500 or greater, 2000 orgreater, 2500 or greater, 3000 or greater, 3500 or greater, 4000 orgreater, or 4500 or greater). In some examples, the compounds of FormulaI can have a molecular weight of 5000 or less (e.g., 4500 or less, 4000or less, 3500 or less, 3000 or less, 2500 or less, 2000 or less, 1500 orless, or 1000 or less). In some examples, the compounds of Formula I canhave a molecular weight of 500 to 5000 (e.g., 500-2500, 500-1500,500-1000, 1000-1500, 1500-2500, 1500-2000, 2000-2500, 2500-5000,2500-3000, 3000-3500, 3500-4000, 4000-4500, 4500-5000, 2500-3500,3500-4500, 500-2000, or 1000-2000).

X_(m) and X_(n)

X_(m) and X_(n) can independently comprise a sequence of 1-10 aminoacids. In some examples, X_(m) and X_(n) can independently comprise 1 ormore amino acid (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, or 9 or more). In some examples, X_(m) andX_(n) can independently comprise 10 or less amino acids (e.g., 9 orless, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less,or 2 or less). In some examples, X_(m) and X_(n) can independentlycomprise 1-10 amino acids (e.g., 1-5, 5-10, 1-3, 3-5, 5-8, 8-10, 1-8,2-6, or 3-7 amino acids). Each amino acid can be a natural ornon-natural amino acid. The term “non-natural amino acid” refers to anorganic compound that is a congener of a natural amino acid in that ithas a structure similar to a natural amino acid so that it mimics thestructure and reactivity of a natural amino acid. The non-natural aminoacid can be a modified amino acid, and/or amino acid analog, that is notone of the 20 common naturally occurring amino acids or the rare naturalamino acids selenocysteine or pyrrolysine. Examples of suitable aminoacids include, but are not limited to, alanine, allosoleucine, arginine,asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine,histidine, isoleucine, leucine, lysine, methionine, napthylalanine,phenylalanine, proline, pyroglutamic acid, serine, threonine,tryptophan, tyrosine, valine, a derivative, or combinations thereof.These are listed in the Table 1 along with their abbreviations usedherein.

TABLE 1 Amino Acid Abbreviations Amino Acid Abbreviations* alanine Ala(A) allosoleucine AIle arginine Arg (R) asparagine Asn (N) aspartic acidAsp (D) cysteine Cys (C) glutamic acid Glu (E) glutamine Gln (Q) glycineGly (G) histidine His (H) isoleucine Ile (I) leucine Leu (L) lysine Lys(K) Methionine Met (M) napthylalanine Nal(Φ) Norleucine Nle (Ω)phenylalanine Phe (F) proline Pro (P) selenocysteine Sec (U) serine Ser(S) threonine Thr (T) tyrosine Tyr (Y) tryptophan Trp (W) valine Val (V)Phenylglycine Phg Propargylglycine Pra

Further, non-natural amino acids and D-amino acids can be used herein.The disclosed methods and compositions are particularly well suited forincorporating non-natural and D-amino acids.

The amino acids can be coupled by a peptide bond. The amino acids can becoupled to each other or to B¹, L¹ or D at the amino group, thecarboxylate group, or the side chain.

In some examples of Formula I, X_(m) and X_(n) can independentlyselected from any of the sequences listed in Table 2.

TABLE 2 Example sequences for X_(m) and X_(n). SEQ ID NO SequenceAbbreviation Phe-Tyr-Ala FYA  1 His-Lys-Gly-Phe-Tyr HKGFY  2Ala-Phe-Trp-Thr-Glu AFWTG  3 His-Ala-Leu-Nle HAL-Nle  4Phg-Tyr-Ala-Lys-Tyr- Phg-YAKYFGKH-Dap Phe-Gly-Lys-His-Dap  5Ala-Phe-Trp-Thr-Glu- AFWTEK-Nle-LAH-Dap Lys-Nle-Leu-Ala-His-Dap  6Phe-Dap-Ser-Val-Pro-Tyr- F-Dap-SVPYH-Dap His-Dap  7 W-F-D-K-F-N-H-DapWFDKFNH-Dap  8 dNal-S-Q-Nal-K-F-R-V-R-Dap dΦ-SQ-dNaI-KFRVR-Dap  9R-R-Nal-R-fF-K-F-dQ-G-Dap RRdΦ-R-fF-KFQG-Dap 10 O-R-Nal-R-fF-K-F-Q-G-DapOR-dΦ-R-fF-KFQG-Dap 11 R-dF-Z-Z-F-K RFZZFK 12 R-D-Phg-Z-N-K RD-Phg-ZNK13 Z-Z-P-G-A-K ZZPGAK 14 Z-Z-A-S-A-K ZZASAK 15 Z-Z-L-P-dT-K ZZLPTK 16Phg-R-N-Z-I-K Phg-RNZIK 17 Z-T-E-A-N-K ZTEANK 18 Z-Nal-V-G-Q-K Z-dΦ-VGQK19 Z-Phg-S-Z-Z-K Z-Phg-SZZK 20 Z-Phg-M-S-Z-K Z-Phg-MSZK 21 Z-S-M-Z-G-KZSMZGK 22 Z-S-Phg-Z-Z-K ZS-Phg-ZZK 23 Z-R-V-D-A-K ZRVDAK 24Arg-Asp-Phg-Pra-Asn RD-Phg-Pra-N 25 FNalR₄-Dap ΦRRRR-Dap 26Phg-Arg-Asn-Pra-Ile Phg-RN-Pra-I 27 Pra-Ser-Phg-Lys-Lys Pra-S-Phg-KK 28Pra-Arg-Val-Asp-Ala Pra-RVDA 29 Ala-Phg-Arg-Asn-Pra-Ile A-Phg-RN-Pra-I30 Phg-Arg-Asn-Pra-Ile-Ala Phg-RN-Pra-IA 31 Ala-Phg-Arg-Asn-Pra-Ile-A-Phg-RN-Pra-IA Ala 32 Ala-Ala-Phg-Arg-Asn-Pra- AA-Phg-RN-Pra-IA Ile-Ala33 A-F-Phg-R-N-Pra-I-A AF-Phg-RN-Pra-I-A 34 A-Abu-Phg-R-N-Pra-I-AbuA-Abu-Phg-RN-Pra- I-Abu 35 Phg-I-Phg-R-N-Pra-I-Abu-K Phg-I-Phg-RN-Pra-I-Abu-K 36 Phg-Phg-R-N-Pra-I-Abu Phg-Phg-RN-Pra-I- Abu 37A-dL-Phg-R-N-Pra-I-D AL-Phg-RN-Pra-ID 38 A-Q-Phg-R-N-Pra-I-DAQ-Phg-RN-Pra-ID 39 I-E-Phg-R-N-Pra-I-D IE-Phg-RN-Pra-ID 40A-S-Phg-R-N-Pra-I-E AS-Phg-RN-Pra-IE 41 L-Phg-R-N-Pra-I-EL-Phg-RN-Pra-IE 42 A-Phg-Phg-R-N-Pra-I-F A-Phg-Phg-RN-Pra- IF 43A-Om-Phg-R-N-Pra-I-F A-Om-Phg-RN-Pra- IF 44 A-Abu-Phg-R-N-Pra-I-NA-Abu-Phg-RN-Pra- IN 45 A-A-Phg-R-N-Pra-I-N dA-A-Phg-RN-Pra- IN 46Phg-N-Phg-R-N-Pra-I-I Phg-N-Phg-RN-Pra- II 47 A-Abu-Phg-R-N-Pra-I-NleA-Abu-Phg-RN-Pra- I-Nle 48 W-Phg-R-N-Pra-I-Phg W-Phg-RN-Pra-I- Phg 49A-N-Phg-R-N-Pra-I-R AN-Phg-RN-Pra-IR 50 R-Nle-Phg-R-N-Pra-I-SR-Nle-Phg-RN-Pra- IS 51 H-Phg-R-N-Pra-I-Y-K-FNal H-Phg-RN-Pra-IYK- Nal52 Ala-Abu-Phg-Arg-Asn-Pra- A-Abu-Phg-RN-Pra- Ile-Abu I-Abu 53Phg-Ile-Phg-Arg-Asn-Pra- Phg-I-Phg-RN-Pra- Ile-Abu I-Abu 54Ala-Leu-Phg-Arg-Asn-Pra- AL-Phg-RN-Pra-ID Ile-Asp 55Ala-Gln-Phg-Arg-Asn-Pra- AQ-Phg-RN-Pra-ID Ile-Asp 56Ala-Orn-Phg-Arg-Asn-Pra- A-Orn-Phg-RN-Pra-IF Ile-Phe 57Ala-Phg-Phg-Arg-Asn-Pra- A-Phg-Phg-RN-Pra-IF Ile-Phe 58Ala-Abu-Phg-Arg-Asn-Pra- A-Abu-Phg-RN-Pra-I- Ile-Abu Abu 59Ala-Ala-Phg-Arg-Asn-Pra- AA-Phg-RN-Pra-IA Ile-Ala 60Ala-Ala-Phe-Arg-Asn-Pra- AAFRN-Pra-IA Ile-Ala 61Ala-Leu-Phe-Arg-Asn-Pra- ALFRN-Pra-ID Ile-Asp 62Phg-Tyr-Ala-Lys-Tyr-Phe- Phg-YAKYFGKH Gly-Lys-His 63Ala-Phe-Trp-Thr-Glu-Lys- AFWTEK-Nle-LAH Nle-Leu-Ala-His

In some examples, X_(m) can by any of SEQ ID NO:1 to SEQ ID NO:63. Insome examples, X_(n) can by any of SEQ ID NO:1 to SEQ ID NO:63. In someexamples, X_(m) can be a variant of any of SEQ ID NO: 1 to SEQ ID NO:25.In some examples, X_(n) can be a variant of any of SEQ ID NO: 1 to SEQID NO:25. Peptide variants are well understood to those of skill in theart and can involve amino acid sequence modifications. For example,amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional, or deletional variants.Insertions include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of 1 to 3 residues.Deletions are characterized by the removal of one or more amino acidresidues from the peptide sequence. Typically, no more than from 1 to 3residues are deleted at any one site within the peptide. Amino acidsubstitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 3 amino acid residues; and deletions will rangeabout from 1 to 3 residues. Deletions or insertions preferably are madein adjacent pairs, i.e. a deletion of 2 residues or insertion of 2residues. Substitutions, deletions, insertions or any combinationthereof can be combined to arrive at a final construct. Substitutionalvariants are those in which at least one residue has been removed and adifferent residue inserted in its place. Such substitutions generallyare made in accordance with the following Table 3 and are referred to asconservative substitutions.

TABLE 3 Amino Acid Substitutions Exemplary Conservative SubstitutionsAla replaced by Ser Leu replaced by Ile or Val Arg replaced by Lys orGln Lys replaced by Arg or Gln Asn replaced by Gln or His Met replacedby Leu or Ile Asp replaced by Glu Phe replaced by Met, Leu, Nal, Phg, orTyr Cys replaced by Ser Ser replaced by Thr Gln replaced by Asn or LysThr replaced by Ser Glu replaced by Asp Trp replaced by Tyr Gly replacedby Pro Tyr replaced by Trp or Phe His replaced by Asn or Gln Valreplaced by Ile or Leu Ile replaced by Leu or Val

Substantial changes in function are made by selecting substitutions thatare less conservative than those in Table 3, i.e., selecting residuesthat differ more significantly in their effect on maintaining (a) thestructure of the peptide backbone in the area of the substitution, forexample as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site or (c) the bulk of theside chain. The substitutions which in general are expected to producethe greatest changes in the protein properties will be those in which(a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g., lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the peptidesprovided herein.

It is understood that one way to define the variants of X_(m) and X_(n)is through defining the variants in terms of homology/identity tospecific known sequences. For example, SEQ ID NO:1 to SEQ ID NO:63 eachsets forth a particular sequence. Specifically disclosed are variants ofthese peptide that have at least, 85%, 90%, 95%, or 97% homology to SEQID NO:1 to SEQ ID NO:63. Those of skill in the art readily understandhow to determine the homology of two proteins. For example, the homologycan be calculated after aligning the two sequences so that the homologyis at its highest level.

In addition to variants of SEQ ID NO:1 to SEQ ID NO:63 are derivativesof these peptides which also function in the disclosed methods andcompositions. Derivatives are formed by replacing one or more residueswith a modified residue, where the side chain of the residue has beenmodified.

Specific Examples

In some examples of Formula I, A comprises benzene. In some examples ofFormula I, B₁ comprises NH. In some examples of Formula I, B₂ comprisesNH. In some examples of Formula I, B₃ comprises NH. In some examples ofFormula I, B₁, B₂, and B₃ each comprise NH. In some examples of FormulaI, p, q, and r are each 0.

In some examples of Formula I, the compounds are of Formula I-A:

wherein X_(m) and X_(n) are as defined in Formula I; R⁰ is selected fromhydrogen, substituted or unsubstituted C₁-C₆ alkyl, or linker to solidphase support; and K has a structure represented by a formula:

In some examples of Formula I-A, R⁰ is hydrogen. In some examples ofFormula I-A, R⁰ is a substituted or unsubstituted C₁-C₆ alkyl. In someexamples of Formula I-A, R⁰ is a linker to a solid phase support.

In some examples of Formula I-A, X_(m) comprises a peptide chain of 3-7amino acid residues. In some examples of Formula I-A, X_(m) comprises apeptide chain of 4-6 amino acid residues. In some examples of FormulaI-A, X_(m) comprises a peptide chain of 3 amino acid residues. In someexamples of Formula I-A, X_(m) comprises a peptide chain of 4 amino acidresidues. In some examples of Formula I-A, X_(m) comprises a peptidechain of 5 amino acid residues. In some examples of Formula I-A, X_(m)comprises a peptide chain of 6 amino acid residues. In some examples ofFormula I-A, X_(m) comprises a peptide chain of 7 amino acid residues.

In some examples of Formula I-A, X_(m) comprises all natural aminoacids.

In some examples of Formula I-A, X_(n) comprises a peptide chain of 2-6amino acid residues. In some examples of Formula I-A, X_(n) comprises apeptide chain of 3-5 amino acid residues. In some examples of FormulaI-A, X_(n) comprises a peptide chain of 2 amino acid residues. In someexamples of Formula I-A, X_(n) comprises a peptide chain of 3 amino acidresidues. In some examples of Formula I-A, X_(n) comprises a peptidechain of 4 amino acid residues. In some examples of Formula I-A, X_(n)comprises a peptide chain of 5 amino acid residues. In some examples ofFormula I-A, X_(n) comprises a peptide chain of 6 amino acid residues.

In some examples of Formula I-A, X_(n) comprises all natural aminoacids.

In some examples of Formula I-A, X_(m) and X_(n) comprise differentamino acid sequences.

In some examples of Formula I-A, the compound has a structurerepresented by Formula I-A-1.

In some examples of Formula I-A, the compound has a structurerepresented by Formula I-A-2.

In some examples of Formula I, A comprises N. In some examples ofFormula I, p, q, and r are each 1.

In some examples of Formula I, the compounds are of Formula I-B:

wherein X_(m), X_(n), L¹, L², B¹, B², B³ and D are as defined in FormulaI.

In some examples of Formula I-B, X_(m) comprises a peptide chain of 3-7amino acid residues. In some examples of Formula I-B, X_(m) comprises apeptide chain of 4-6 amino acid residues. In some examples of FormulaI-B, X_(m) comprises a peptide chain of 3 amino acid residues. In someexamples of Formula I-B, X_(m) comprises a peptide chain of 4 amino acidresidues. In some examples of Formula I-B, X_(m) comprises a peptidechain of 5 amino acid residues. In some examples of Formula I-B, X_(m)comprises a peptide chain of 6 amino acid residues. In some examples ofFormula I-B, X_(m) comprises a peptide chain of 7 amino acid residues.

In some examples of Formula I-B, X_(m) comprises all natural aminoacids.

In some examples of Formula I-B, X_(n) comprises a peptide chain of 2-6amino acid residues. In some examples of Formula I-B, X_(n) comprises apeptide chain of 3-5 amino acid residues. In some examples of FormulaI-B, X_(n) comprises a peptide chain of 2 amino acid residues. In someexamples of Formula I-B, X_(n) comprises a peptide chain of 3 amino acidresidues. In some examples of Formula I-B, X_(n) comprises a peptidechain of 4 amino acid residues. In some examples of Formula I-B, X_(n)comprises a peptide chain of 5 amino acid residues. In some examples ofFormula I-B, X_(n) comprises a peptide chain of 6 amino acid residues.

In some examples of Formula I-B, X_(n) comprises all natural aminoacids.

In some examples of Formula I-B, X_(m) and X_(n) comprise differentamino acid sequences.

Also disclosed herein are compounds comprising a residue of trimesicacid, the residue bearing three carboxyl functionalities; a lysineresidue covalently bonded to the first carboxyl functionality of thetrimesic acid residue; a first peptide chain of 1-10 amino acidresidues, X_(m), covalently bonded to the second carboxyl functionalityof the trimesic acid residue and to the lysine residue; and a secondpeptide chain of 1-10 amino acid residues, X_(n), covalently bonded tothe third carboxyl functionality of the trimesic acid residue and to thelysine residue.

In some examples, the trimesic acid based compound is covalently linkedto a solid phase support.

In some examples of the trimesic acid based compounds, the first peptidechain comprises a peptide chain of 3-7 amino acid residues. In someexamples of the trimesic acid based compounds, the first peptide chaincomprises a peptide chain of 4-6 amino acid residues. In some examplesof the trimesic acid based compounds, the first peptide chain comprisesa peptide chain of 3 amino acid residues. In some examples of thetrimesic acid based compounds, the first peptide chain comprises apeptide chain of 4 amino acid residues. In some examples of the trimesicacid based compounds, the first peptide chain comprises a peptide chainof 5 amino acid residues. In some examples of the trimesic acid basedcompounds, the first peptide chain comprises a peptide chain of 6 aminoacid residues. In some examples of the trimesic acid based compounds,the first peptide chain comprises a peptide chain of 7 amino acidresidues.

In some examples of the trimesic acid based compounds, the first peptidechain comprises all natural amino acids.

In some examples of the trimesic acid based compounds, the secondpeptide chain comprises a peptide chain of 2-6 amino acid residues. Insome examples of the trimesic acid based compounds, the second peptidechain comprises a peptide chain of 3-5 amino acid residues. In someexamples of the trimesic acid based compounds, the second peptide chaincomprises a peptide chain of 2 amino acid residues. In some examples ofthe trimesic acid based compounds, the second peptide chain comprises apeptide chain of 3 amino acid residues. In some examples of the trimesicacid based compounds, the second peptide chain comprises a peptide chainof 4 amino acid residues. In some examples of the trimesic acid basedcompounds, the second peptide chain comprises a peptide chain of 5 aminoacid residues. In some examples of the trimesic acid based compounds,the second peptide chain comprises a peptide chain of 6 amino acidresidues.

In some examples of the trimesic acid based compounds, the first peptidechain and the second peptide chain comprise different amino acidsequences.

In some examples of the trimesic acid based compounds, the secondpeptide chain comprises all natural amino acids.

Also disclosed herein is a library comprising a plurality of thedisclosed compounds. In some examples, the compounds in the library arecovalently linked to a solid phase support.

In some examples, the compounds in the library and/or the solid phasesupport bear a label moiety. The label moiety can comprise anydetectable label. Examples of suitable detectable labels include, butare not limited to, a UV-Vis label, a near-infrared label, a luminescentgroup, a phosphorescent group, a magnetic spin resonance label, aphotosensitizer, a photocleavable moiety, a chelating center, a heavyatom, a radioactive isotope, a isotope detectable spin resonance label,a paramagnetic moiety, a chromophore, or any combination thereof. Insome embodiments, the label is detectable without the addition offurther reagents.

In some embodiments, the label moiety is a biocompatible label moiety,such that the compounds can be suitable for use in a variety ofbiological applications. “Biocompatible” and “biologically compatible”,as used herein, generally refer to compounds that are, along with anymetabolites or degradation products thereof, generally non-toxic tocells and tissues, and which do not cause any significant adverseeffects to cells and tissues when cells and tissues are incubated (e.g.,cultured) in their presence.

The label moiety can contain a luminophore such as a fluorescent labelor near-infrared label. Examples of suitable luminophores include, butare not limited to, metal porphyrins; benzoporphyrins;azabenzoporphyrine; napthoporphyrin; phthalocyanine; polycyclic aromatichydrocarbons such as perylene, perylene diimine, pyrenes; azo dyes;xanthene dyes; boron dipyoromethene, aza-boron dipyoromethene, cyaninedyes, metal-ligand complex such as bipyridine, bipyridyls,phenanthroline, coumarin, and acetylacetonates of ruthenium and iridium;acridine, oxazine derivatives such as benzophenoxazine; aza-annulene,squaraine; 8-hydroxyquinoline, polymethines, luminescent producingnanoparticle, such as quantum dots, nanocrystals; carbostyril; terbiumcomplex; inorganic phosphor; ionophore such as crown ethers affiliatedor derivatized dyes; or combinations thereof. Specific examples ofsuitable luminophores include, but are not limited to, Pd (II)octaethylporphyrin; Pt (II)octaethylporphyrin; Pd (II)tetraphenylporphyrin; Pt (II) tetraphenylporphyrin; Pd (II)mesotetraphenylporphyrin tetrabenzoporphine; Pt (II) meso-tetraphenymetrylbenzoporphyrin; Pd (II) octaethylporphyrin ketone; Pt (II)octaethylporphyrin ketone; Pd (II)mesotetra(pentafluorophenyl)porphyrin; Pt (II) meso-tetra(pentafluorophenyl) porphyrin; Ru (II)tris(4,7-diphenyl-1,10-phenanthroline) (Ru (dpp)₃); Ru (II)tris(1,10-phenanthroline) (Ru(phen)₃), tris(2,2′-bipyridine)rutheniumrn(II) chloride hexahydrate (Ru(bpy)₃); erythrosine B; fluorescein;fluorescein isothiocyanate (FITC); eosin; iridium (III)((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin)); indium (III)((benzothiazol-2-yl)-7-(diethylamino)coumarin))-2-(acetylacetonate);Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow;Texas Red; rhodamine B; rhodamine 6G; sulfur rhodamine; m-cresol; thymolblue; xylenol blue; cresol red; chlorophenol blue; bromocresol green;bromcresol red; bromothymol blue; Cy2; a Cy3; a Cy5; a Cy5.5; Cy7;4-nitirophenol; alizarin; phenolphthalein; ocresolphthalein;chlorophenol red; calmagite; bromo-xylenol; phenol red; neutral red;nitrazine; 3,4,5,6-tetrabromphenolphtalein; congo red; fluorescein;eosin; 2′,7′-dichlorofluorescein; 5(6)carboxy-fluorecsein;carboxynaphtofluorescein; 8-hydroxypyrene-1,3,6-trisulfonic acid;seminaphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride;(4,7-diphenyl-1,10-phenanthroline) ruthenium (II) tetraphenylboron;platinum (II) octaethylporphyin; dialkylcarbocyanine;dioctadecylcycloxacarbocyanine; fluorenylmethyloxycarbonyl chloride;7-amino-4-methylcourmarin (Amc); green fluorescent protein (GFP); andderivatives or combinations thereof.

In some examples, the label moiety is a fluorescence label. In someexamples, the fluorescence label is a small molecule. Such smallmolecules are known in the art.

In some examples, the label moiety has a structure represented by aformula:

Use of Compounds

In some examples, the disclosed compounds can be administered to asubject. In some examples, the animal is a mammal. In some examples, themammal is a human. In some examples, the mammal is a mouse. In someexamples, the mammal is a rodent. In some examples, the animal is a fishor a bird.

In some examples, the disclosed compounds treat or prevent cancer whenadministered at a dose of greater than about 1 mg per day in a human. Insome examples, the disclosed compounds treat or prevent cancer whenadministered at a dose of greater than about 5 mg per day in a human. Insome examples, the disclosed compounds treat or prevent cancer whenadministered at a dose of greater than about 10 mg per day in a human.In some examples In some examples, the disclosed compounds treat orprevent a cancer when administered at a dose of greater than about 50 mgper day in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at a dose of greater than about 75 mgper day in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at a dose of greater than about 100 mgper day in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at a dose of greater than about 150 mgper day in a human. In some examples, the disclosed treat or preventcancer when administered at a dose of greater than about 200 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 250 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 300 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 400 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 500 mg per dayin a human In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 750 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at a dose of greater than about 1000 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at an amount of greater than about 1500 mg perday in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at a dose of greater than about 2000 mgper day in a human.

In some examples, the disclosed compounds treat or prevent cancer whenadministered at an oral dose of greater than about 5 mg per day in ahuman. In some examples, the disclosed compounds treat or prevent cancerwhen administered at an oral dose of greater than about 10 mg per day ina human. In some examples, the disclosed compounds treat or preventcancer when administered at an oral dose of greater than about 25 mg perday in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at an oral dose of greater than about50 mg per day in a human. In some examples, the disclosed compoundstreat or prevent cancer when administered at an oral dose of greaterthan about 75 mg per day in a human. In some examples, the disclosedcompounds treat or prevent cancer when administered at an oral dose ofgreater than about 100 mg per day in a human. In some examples, thedisclosed compounds treat or prevent cancer when administered at an oraldose of greater than about 150 mg per day in a human. In some examples,the disclosed treat or prevent cancer when administered at an oral doseof greater than about 200 mg per day in a human. In some examples, thedisclosed compounds treat or prevent cancer when administered at an oraldose of greater than about 250 mg per day in a human. In some examples,the disclosed compounds treat or prevent cancer when administered at anoral dose of greater than about 300 mg per day in a human. In someexamples, the disclosed compounds treat or prevent cancer whenadministered at an oral dose of greater than about 400 mg per day in ahuman. In some examples, the disclosed compounds treat or prevent cancerwhen administered at an oral dose of greater than about 500 mg per dayin a human. In some examples, the disclosed compounds treat or preventcancer when administered at an oral dose of greater than about 750 mgper day in a human. In some examples, the disclosed compounds treat orprevent cancer when administered at an oral dose of greater than about1000 mg per day in a human. In some examples, the disclosed compoundstreat or prevent cancer when administered at an oral dose of greaterthan about 1500 mg per day in a human. In some examples, the disclosedcompounds treat or prevent cancer when administered at an oral dose ofgreater than about 2000 mg per day in a human.

Pharmaceutical Compositions

Also disclosed herein are pharmaceutical compositions comprising thedisclosed compounds. That is, a pharmaceutical composition can beprovided comprising a therapeutically effective amount of at least onedisclosed compound. In some examples, a pharmaceutical composition canbe provided comprising a prophylactically effective amount of at leastone disclosed compound.

Also disclosed herein are pharmaceutical compositions comprising apharmaceutically acceptable carrier and any of the compounds disclosedherein, wherein the compound is present in an effective amount. Alsodisclosed herein are neutraceutical compositions comprising aneutraceutically acceptable carrier and any of the compounds disclosedherein, wherein the compound is present in an effective amount.

In some examples of the compositions, the compound is present in anamount greater than about an amount selected from 5 mg, 10 mg, 25 mg, 50mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400, mg, 500 mg, 750 mg,1000 mg, 1,500 mg, or 2,000 mg.

The disclosed pharmaceutical compositions can further comprise one ormore anticancer drugs.

Example anticancer drugs include 13-cis-Retinoic Acid,2-Amino-6-Mercaptopurine, 2-CdA, 2-Chlorodeoxyadenosine, 5-fluorouracil,6-Thioguanine, 6-Mercaptopurine, Accutane, Actinomycin-D, Adriamycin,Adrucil, Agrylin, Ala-Cort, Aldesleukin, Alemtuzumab, Alitretinoin,Alkaban-AQ, Alkeran, All-transretinoic acid, Alpha interferon,Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide,Anandron, Anastrozole, Arabinosylcytosine, Aranesp, Aredia, Arimidex,Aromasin, Arsenic trioxide, Asparaginase, ATRA, Avastin, BCG, BCNU,Bevacizumab, Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin,Bortezomib, Busulfan, Busulfex, C225, Calcium Leucovorin, Campath,Camptosar, Camptothecin-11, Capecitabine, Carac, Carboplatin,Carmustine, Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine,cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine,Cortisone, Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine,Cytarabine liposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin,Darbepoetin alfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride,Daunorubicin liposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone,Denileukin diftitox, DepoCyt, Dexamethasone, Dexamethasone acetate,Dexamethasone sodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC,Diodex, Docetaxel, Doxil, Doxorubicin, Doxorubicin liposomal, Droxia,DTIC, DTIC-Dome, Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar,Emcyt, Epirubicin, Epoetin alfa, Erbitux, Erwinia L-asparaginase,Estramustine, Ethyol, Etopophos, Etoposide, Etoposide phosphate,Eulexin, Evista, Exemestane, Fareston, Faslodex, Femara, Filgrastim,Floxuridine, Fludara, Fludarabine, Fluoroplex, Fluorouracil,Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR,Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin,Gemzar, Gleevec, Lupron, Lupron Depot, Matulane, Maxidex,Mechlorethamine, -Mechlorethamine Hydrochlorine, Medralone, Medrol,Megace, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex, Methotrexate, Methotrexate Sodium, Methylprednisolone, Mylocel,Letrozole, Neosar, Neulasta, Neumega, Neupogen, Nilandron, Nilutamide,Nitrogen Mustard, Novaldex, Novantrone, Octreotide, Octreotide acetate,Oncospar, Oncovin, Ontak, Onxal, Oprevelkin, Orapred, Orasone,Oxaliplatin, Paclitaxel, Pamidronate, Panretin, Paraplatin, Pediapred,PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRON,PEG-L-asparaginase, Phenylalanine Mustard, Platinol, PlatinolAQ,Prednisolone, Prednisone, Prelone, Procarbazine, PROCRIT, Proleukin,Prolifeprospan 20 with Carmustine implant, Purinethol, Raloxifene,Rheumatrex, Rituxan, Rituximab, Roveron-A (interferon alfa-2a), Rubex,Rubidomycin hydrochloride, Sandostatin, Sandostatin LAR, Sargramostim,Solu-Cortef, Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin,Taxol, Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, LPAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR.

In some examples, the pharmaceutical composition is administered to asubject. In some examples, the subject is a mammal, fish or bird. Insome examples, the mammal is a primate. In some examples, the mammal isa human. In some examples, the human is a patient.

In some examples, the pharmaceutical composition is administeredfollowing identification of the mammal in need of treatment of cancer.In some examples, the pharmaceutical composition is administeredfollowing identification of the mammal in need of prevention of cancer.In some examples, the mammal has been diagnosed with a need fortreatment of cancer to the administering step.

In some examples, the disclosed pharmaceutical compositions comprise thedisclosed compounds (including pharmaceutically acceptable salt(s)thereof) as an active ingredient, a pharmaceutically acceptable carrier,and, optionally, other therapeutic ingredients or adjuvants. The instantcompositions include those suitable for oral, rectal, topical, andparenteral (including subcutaneous, intramuscular, and intravenous)administration, although the most suitable route in any given case willdepend on the particular host, and nature and severity of the conditionsfor which the active ingredient is being administered. Thepharmaceutical compositions can be conveniently presented in unit dosageform and prepared by any of the methods well known in the art ofpharmacy.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts prepared from pharmaceutically or neutraceutically acceptablenon-toxic bases or acids. When the compound is acidic, its correspondingsalt can be conveniently prepared from pharmaceutically acceptablenon-toxic bases, including inorganic bases and organic bases. Saltsderived from such inorganic bases include aluminum, ammonium, calcium,copper (-ic and -ous), ferric, ferrous, lithium, magnesium, manganese(-ic and -ous), potassium, sodium, zinc and the like salts. Particularlypreferred are the ammonium, calcium, magnesium, potassium and sodiumsalts. Salts derived from pharmaceutically acceptable organic non-toxicbases include salts of primary, secondary, and tertiary amines, as wellas cyclic amines and substituted amines such as naturally occurring andsynthesized substituted amines. Other pharmaceutically orneutraceutically acceptable organic non-toxic bases from which salts canbe formed include ion exchange resins such as, for example, arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

As used herein, the term “pharmaceutically acceptable non-toxic acids”,includes inorganic acids, organic acids, and salts prepared thereof, forexample, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,p-toluenesulfonic acid and the like. Preferred are citric, hydrobromic,hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

In practice, the compounds disclosed herein, or pharmaceuticallyacceptable salts thereof, or neutraceutically acceptable salts thereof,can be combined as the active ingredient in intimate admixture with apharmaceutical carrier or neutraceutical carrier according toconventional pharmaceutical compounding techniques or conventionalneutraceutical compounding techniques. The carrier can take a widevariety of forms depending on the form of preparation desired foradministration, e.g., oral or parenteral (including intravenous). Thus,the pharmaceutical compositions or neutraceutical compositions disclosedherein can be presented as discrete units suitable for oraladministration such as capsules, cachets or tablets each containing apredetermined amount of the active ingredient. Further, the compositionscan be presented as a powder, as granules, as a solution, as asuspension in an aqueous liquid, as a non-aqueous liquid, as anoil-in-water emulsion or as a water-in-oil liquid emulsion. In additionto the common dosage forms set out above, the compounds disclosedherein, and/or pharmaceutically acceptable salt(s) thereof, can also beadministered by controlled release means and/or delivery devices. Thecompositions can be prepared by any of the methods of pharmacy. Ingeneral, such methods include a step of bringing into association theactive ingredient with the carrier that constitutes one or moreingredients. In general, the compositions are prepared by uniformly andintimately admixing the active ingredient with liquid carriers or finelydivided solid carriers or both. The product can then be convenientlyshaped into the desired presentation.

Thus, the pharmaceutical compositions disclosed herein can include apharmaceutically acceptable carrier and a compound or a pharmaceuticallyacceptable salt of the compounds disclosed herein. The compoundsdisclosed herein, or pharmaceutically acceptable salts thereof, can alsobe included in pharmaceutical compositions in combination with one ormore other therapeutically active compounds.

The pharmaceutical carrier employed can be, for example, a solid,liquid, or gas. Examples of solid carriers include lactose, terra alba,sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, andstearic acid. Examples of liquid carriers are sugar syrup, peanut oil,olive oil, and water. Examples of gaseous carriers include carbondioxide and nitrogen.

In preparing the compositions for oral dosage form, any convenientpharmaceutical media can be employed. For example, water, glycols, oils,alcohols, flavoring agents, preservatives, coloring agents and the likecan be used to form oral liquid preparations such as suspensions,elixirs and solutions; while carriers such as starches, sugars,microcrystalline cellulose, diluents, granulating agents, lubricants,binders, disintegrating agents, and the like can be used to form oralsolid preparations such as powders, capsules and tablets. Because oftheir ease of administration, tablets and capsules can be used for oraldosage units whereby solid pharmaceutical carriers are employed.Optionally, tablets can be coated by standard aqueous or nonaqueoustechniques

A tablet containing any of the compositions disclosed herein can beprepared by compression or molding, optionally with one or moreaccessory ingredients or adjuvants. Compressed tablets can be preparedby compressing, in a suitable machine, the active ingredient in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, surface active or dispersing agent.Molded tablets can be made by molding in a suitable machine, a mixtureof the powdered compound moistened with an inert liquid diluent.

The pharmaceutical compositions disclosed herein can comprise any of thecompounds disclosed herein (or pharmaceutically or neutraceuticallyacceptable salts thereof) as an active ingredient, a pharmaceuticallyacceptable carrier or neutraceutically acceptable carrier, andoptionally one or more additional therapeutic agents or adjuvants. Theinstant compositions include compositions suitable for oral, rectal,topical, and parenteral (including subcutaneous, intramuscular, andintravenous) administration, although the most suitable route in anygiven case will depend on the particular host, and nature and severityof the conditions for which the active ingredient is being administered.The pharmaceutical compositions can be conveniently presented in unitdosage form and prepared by any of the methods well known in the art ofpharmacy.

Pharmaceutical compositions suitable for parenteral administration canbe prepared as solutions or suspensions of the active compounds inwater. A suitable surfactant can be included such as, for example,hydroxypropylcellulose. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, and mixtures thereof in oils. In someexamples, a preservative can be included to prevent the detrimentalgrowth of microorganisms.

Pharmaceutical compositions disclosed herein suitable for injectable useinclude sterile aqueous solutions or dispersions. Furthermore, thecompositions can be in the form of sterile powders for theextemporaneous preparation of such sterile injectable solutions ordispersions. In some examples, the final injectable form can be sterileand can be effectively fluid for easy syringability. In some examples,the pharmaceutical compositions can be stable under the conditions ofmanufacture and storage; thus, they can be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), vegetable oils, and suitable mixtures thereof

Pharmaceutical compositions disclosed herein can be in a form suitablefor topical use such as, for example, an aerosol, cream, ointment,lotion, dusting powder, mouth washes, gargles, and the like. In someexamples, the compositions can be in a form suitable for use intransdermal devices. These formulations can be prepared, utilizing anyof the compounds disclosed herein or pharmaceutically acceptable saltsthereof, via conventional processing methods. As an example, a cream orointment can be prepared by mixing hydrophilic material and water,together with about 5 wt % to about 10 wt % of the compound, to producea cream or ointment having a desired consistency.

Pharmaceutical compositions disclosed herein can be in a form suitablefor rectal administration wherein the carrier is a solid. In someexamples, the mixture forms unit dose suppositories. Suitable carriersinclude cocoa butter and other materials commonly used in the art. Thesuppositories can be conveniently formed by first admixing thecomposition with the softened or melted carriers) followed by chillingand shaping in molds.

In addition to the aforementioned carrier ingredients, thepharmaceutical formulations described above can include, as appropriate,one or more additional carrier ingredients such as diluents, buffers,flavoring agents, binders, surface-active agents, thickeners,lubricants, preservatives (including anti-oxidants) and the like.Furthermore, other adjuvants can be included to render the formulationisotonic with the blood of the intended recipient. Compositionscontaining any of the compounds disclosed herein, and/orpharmaceutically acceptable salts thereof, can also be prepared inpowder or liquid concentrate form.

In the treatment of cancer, the dosage level of the active ingredientcomprising the compound or compositions disclosed herein can be about0.01 to 500 mg per kg patient body weight per day and can beadministered in single or multiple doses. In some examples, he dosagelevel will be about 0.1 to about 250 mg/kg per day; such as 0.5 to 100mg/kg per day. A suitable dosage level can be about 0.01 to 250 mg/kgper day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg perday. Within this range the dosage can be 0.05 to 0.5, 0.5 to 5.0, or 5.0to 50 mg/kg per day. For oral administration, the compositions can be,for example, in the form of tablets containing 1.0 to 1000 milligrams ofthe active ingredient, particularly 1.0, 5.0, 10, 15, 20, 25, 50, 75,100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, and 1000milligrams of the active ingredient for the symptomatic adjustment ofthe dosage of the patient to be treated. The compound can beadministered on a regimen of 1 to 4 times per day, such as, for example,once or twice per day. This dosing regimen can be adjusted to providethe optimal therapeutic response.

It is understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors. Such factorsinclude the age, body weight, general health, sex, and diet of thepatient. Other factors include the time and route of administration,rate of excretion, drug combination, and the type and severity of theparticular disease or infection undergoing therapy.

Also disclosed herein are methods for the manufacture of a medicamentfor treating cancer in mammals (e.g., humans) comprising combining oneor more disclosed compounds, products, or compositions with apharmaceutically acceptable carrier or diluent. In some examples, themethod for manufacturing a medicament comprises combining at least onedisclosed compound or at least one disclosed product with apharmaceutically acceptable carrier or diluent.

The disclosed pharmaceutical compositions can further comprise othertherapeutically active compounds, which are usually applied in thetreatment of the above mentioned pathological conditions.

It is understood that the disclosed compositions can be prepared fromthe disclosed compounds. It is also understood that the disclosedcompositions can be employed in the disclosed methods of using.

Compositions, Formulations and Methods of Administration

In vivo application of the disclosed compounds, and compositionscontaining them, can be accomplished by any suitable method andtechnique presently or prospectively known to those skilled in the art.For example, the disclosed compounds can be formulated in aphysiologically- or pharmaceutically-acceptable form and administered byany suitable route known in the art including, for example, oral, nasal,rectal, topical, and parenteral routes of administration. As usedherein, the term parenteral includes subcutaneous, intradermal,intravenous, intramuscular, intraperitoneal, and intrasternaladministration, such as by injection. Administration of the disclosedcompounds or compositions can be a single administration, or atcontinuous or distinct intervals as can be readily determined by aperson skilled in the art.

The compounds disclosed herein, and compositions comprising them, canalso be administered utilizing liposome technology, slow releasecapsules, implantable pumps, and biodegradable containers. Thesedelivery methods can, advantageously, provide a uniform dosage over anextended period of time. The compounds can also be administered in theirsalt derivative forms or crystalline forms.

The compounds disclosed herein can be formulated according to knownmethods for preparing pharmaceutically acceptable compositions.Formulations are described in detail in a number of sources which arewell known and readily available to those skilled in the art. Forexample, Remington's Pharmaceutical Science by E. W. Martin (1995)describes formulations that can be used in connection with the disclosedmethods. In general, the compounds disclosed herein can be formulatedsuch that an effective amount of the compound is combined with asuitable carrier in order to facilitate effective administration of thecompound. The compositions used can also be in a variety of forms. Theseinclude, for example, solid, semi-solid, and liquid dosage forms, suchas tablets, pills, powders, liquid solutions or suspension,suppositories, injectable and infusible solutions, and sprays. Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions also preferably includeconventional pharmaceutically-acceptable carriers and diluents which areknown to those skilled in the art. Examples of carriers or diluents foruse with the compounds include ethanol, dimethyl sulfoxide, glycerol,alumina, starch, saline, and equivalent carriers and diluents. Toprovide for the administration of such dosages for the desiredtherapeutic treatment, compositions disclosed herein can advantageouslycomprise between about 0.1% and 100% by weight of the total of one ormore of the subject compounds based on the weight of the totalcomposition including carrier or diluent.

Formulations suitable for administration include, for example, aqueoussterile injection solutions, which can contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions, which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and can be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions can be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions disclosed herein can include other agents conventional inthe art having regard to the type of formulation in question.

Compounds disclosed herein, and compositions comprising them, can bedelivered to a cell either through direct contact with the cell or via acarrier means. Carrier means for delivering compounds and compositionsto cells are known in the art and include, for example, encapsulatingthe composition in a liposome moiety. Another means for delivery ofcompounds and compositions disclosed herein to a cell comprisesattaching the compounds to a protein or nucleic acid that is targetedfor delivery to the target cell. U.S. Pat. No. 6,960,648 and U.S.Application Publication Nos. 20030032594 and 20020120100 disclose aminoacid sequences that can be coupled to another composition and thatallows the composition to be translocated across biological membranes.U.S. Application Publication No. 20020035243 also describes compositionsfor transporting biological moieties across cell membranes forintracellular delivery. Compounds can also be incorporated intopolymers, examples of which include poly (D-L lactide-co-glycolide)polymer for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] in a 20:80 molar ratio (as used in GLIADEL);chondroitin; chitin; and chitosan.

For the treatment of oncological disorders, the compounds disclosedherein can be administered to a patient in need of treatment incombination with other antitumor or anticancer substances and/or withradiation and/or photodynamic therapy and/or with surgical treatment toremove a tumor. These other substances or treatments can be given at thesame as or at different times from the compounds disclosed herein. Forexample, the compounds disclosed herein can be used in combination withmitotic inhibitors such as taxol or vinblastine, alkylating agents suchas cyclophosamide or ifosfamide, antimetabolites such as 5-fluorouracilor hydroxyurea, DNA intercalators such as adriamycin or bleomycin,topoisomerase inhibitors such as etoposide or camptothecin,antiangiogenic agents such as angiostatin, antiestrogens such astamoxifen, and/or other anti-cancer drugs or antibodies, such as, forexample, GLEEVEC (Novartis Pharmaceuticals Corporation) and HERCEPTIN(Genentech, Inc.), respectively, or an immunotherapeutic such asipilimumab and bortezomib.

In certain examples, compounds and compositions disclosed herein can belocally administered at one or more anatomical sites, such as sites ofunwanted cell growth (such as a tumor site or benign skin growth, e.g.,injected or topically applied to the tumor or skin growth), optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent. Compounds and compositions disclosed herein can besystemically administered, such as intravenously or orally, optionallyin combination with a pharmaceutically acceptable carrier such as aninert diluent, or an assimilable edible carrier for oral delivery. Theycan be enclosed in hard or soft shell gelatin capsules, can becompressed into tablets, or can be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, the activecompound can be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like can also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring can be added. Whenthe unit dosage form is a capsule, it can contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials can be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules can be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir cancontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound canbe incorporated into sustained-release preparations and devices.

Compounds and compositions disclosed herein, including pharmaceuticallyacceptable salts or prodrugs thereof, can be administered intravenously,intramuscularly, or intraperitoneally by infusion or injection.Solutions of the active agent or its salts can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient, which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various other antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion of agents that delay absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating a compoundand/or agent disclosed herein in the required amount in the appropriatesolvent with various other ingredients enumerated above, as required,followed by filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, compounds and agents disclosed herein can beapplied in as a liquid or solid. However, it will generally be desirableto administer them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which can be a solid or aliquid. Compounds and agents and compositions disclosed herein can beapplied topically to a subject's skin to reduce the size (and caninclude complete removal) of malignant or benign growths, or to treat aninfection site. Compounds and agents disclosed herein can be applieddirectly to the growth or infection site. Preferably, the compounds andagents are applied to the growth or infection site in a formulation suchas an ointment, cream, lotion, solution, tincture, or the like.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Useful dosages of the compounds and agents and pharmaceuticalcompositions disclosed herein can be determined by comparing their invitro activity, and in vivo activity in animal models. Methods for theextrapolation of effective dosages in mice, and other animals, to humansare known to the art.

The dosage ranges for the administration of the compositions are thoselarge enough to produce the desired effect in which the symptoms ordisorder are affected. The dosage should not be so large as to causeadverse side effects, such as unwanted cross-reactions, anaphylacticreactions, and the like. Generally, the dosage will vary with the age,condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can be adjusted by theindividual physician in the event of any counter indications. Dosage canvary, and can be administered in one or more dose administrations daily,for one or several days.

Also disclosed are pharmaceutical compositions that comprise a compounddisclosed herein in combination with a pharmaceutically acceptablecarrier. In some examples, the pharmaceutical compositions are adaptedfor oral, topical or parenteral administration. The dose administered toa patient, particularly a human, should be sufficient to achieve atherapeutic response in the patient over a reasonable time frame,without lethal toxicity, and preferably causing no more than anacceptable level of side effects or morbidity. One skilled in the artwill recognize that dosage will depend upon a variety of factorsincluding the condition (health) of the subject, the body weight of thesubject, kind of concurrent treatment, if any, frequency of treatment,therapeutic ratio, as well as the severity and stage of the pathologicalcondition.

Methods of Using the Compounds and Compositions

Also provided herein are methods of use of the compounds or compositionsdescribed herein. Also provided herein are methods for treating adisease or pathology in a subject in need thereof comprisingadministering to the subject an effective amount of any of the compoundsor compositions described herein.

A very important application is for specific delivery of drugs such asanticancer drugs. The bicyclic peptides disclosed herein can be directedto a cancer-specific or overexpressed surface protein. Then ananticancer drug can be covalently or noncovaelently attached to thebicyclic peptide.

Also provided herein are methods of treating, preventing, orameliorating cancer in a subject. The methods include administering to asubject an effective amount of one or more of the compounds orcompositions described herein, or a pharmaceutically acceptable saltthereof. The compounds and compositions described herein orpharmaceutically acceptable salts thereof are useful for treating cancerin humans, e.g., pediatric and geriatric populations, and in animals,e.g., veterinary applications. The disclosed methods can optionallyinclude identifying a patient who is or can be in need of treatment of acancer. Examples of cancer types treatable by the compounds andcompositions described herein include bladder cancer, brain cancer,breast cancer, colorectal cancer, cervical cancer, gastrointestinalcancer, genitourinary cancer, head and neck cancer, lung cancer, ovariancancer, pancreatic cancer, prostate cancer, renal cancer, skin cancer,and testicular cancer. Further examples include cancer and/or tumors ofthe anus, bile duct, bone, bone marrow, bowel (including colon andrectum), eye, gall bladder, kidney, mouth, larynx, esophagus, stomach,testis, cervix, mesothelioma, neuroendocrine, penis, skin, spinal cord,thyroid, vagina, vulva, uterus, liver, muscle, blood cells (includinglymphocytes and other immune system cells). Further examples of cancerstreatable by the compounds and compositions described herein includecarcinomas, Karposi's sarcoma, melanoma, mesothelioma, soft tissuesarcoma, pancreatic cancer, lung cancer, leukemia (acute lymphoblastic,acute myeloid, chronic lymphocytic, chronic myeloid, and other), andlymphoma (Hodgkin's and non-Hodgkin's), and multiple myeloma.

In some examples, the cancer can be associated with TNF-α induced celldeath.

The methods of treatment or prevention of cancer described herein canfurther include treatment with one or more additional agents (e.g., ananti-cancer agent or ionizing radiation). The one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be administered in any order,including simultaneous administration, as well as temporally spacedorder of up to several days apart. The methods can also include morethan a single administration of the one or more additional agents and/orthe compounds and compositions or pharmaceutically acceptable saltsthereof as described herein. The administration of the one or moreadditional agents and the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein can be by the same ordifferent routes. When treating with one or more additional agents, thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be combined into a pharmaceutical compositionthat includes the one or more additional agents.

For example, the compounds or compositions or pharmaceuticallyacceptable salts thereof as described herein can be combined into apharmaceutical composition with an additional anti-cancer agent, such as13-cis-Retinoic Acid, 2-Amino-6-Mercaptopurine, 2-CdA,2-Chlorodeoxyadenosine, 5-fluorouracil, 6-Thioguanine, 6-Mercaptopurine,Accutane, Actinomycin-D, Adriamycin, Adrucil, Agrylin, Ala-Cort,Aldesleukin, Alemtuzumab, Alitretinoin, Alkaban-AQ, Alkeran,All-transretinoic acid, Alpha interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron, Anastrozole,Arabinosylcytosine, Aranesp, Aredia, Arimidex, Aromasin, Arsenictrioxide, Asparaginase, ATRA, Avastin, BCG, BCNU, Bevacizumab,Bexarotene, Bicalutamide, BiCNU, Blenoxane, Bleomycin, Bortezomib,Busulfan, Busulfex, C225, Calcium Leucovorin, Campath, Camptosar,Camptothecin-11, Capecitabine, Carac, Carboplatin, Carmustine,Carmustine wafer, Casodex, CCNU, CDDP, CeeNU, Cerubidine, cetuximab,Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone,Cosmegen, CPT-11, Cyclophosphamide, Cytadren, Cytarabine, Cytarabineliposomal, Cytosar-U, Cytoxan, Dacarbazine, Dactinomycin, Darbepoetinalfa, Daunomycin, Daunorubicin, Daunorubicin hydrochloride, Daunorubicinliposomal, DaunoXome, Decadron, Delta-Cortef, Deltasone, Denileukindiftitox, DepoCyt, Dexamethasone, Dexamethasone acetate, Dexamethasonesodium phosphate, Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel,Doxil, Doxorubicin, Doxorubicin liposomal, Droxia, DTIC, DTIC-Dome,Duralone, Efudex, Eligard, Ellence, Eloxatin, Elspar, Emcyt, Epirubicin,Epoetin alfa, Erbitux, Erwinia L-asparaginase, Estramustine, Ethyol,Etopophos, Etoposide, Etoposide phosphate, Eulexin, Evista, Exemestane,Fareston, Faslodex, Femara, Filgrastim, Floxuridine, Fludara,Fludarabine, Fluoroplex, Fluorouracil, Fluorouracil (cream),Fluoxymesterone, Flutamide, Folinic Acid, FUDR, Fulvestrant, G-CSF,Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, Gleevec, Lupron,Lupron Depot, Matulane, Maxidex, Mechlorethamine, -MechlorethamineHydrochlorine, Medralone, Medrol, Megace, Megestrol, Megestrol Acetate,Melphalan, Mercaptopurine, Mesna, Mesnex, Methotrexate, MethotrexateSodium, Methylprednisolone, Mylocel, Letrozole, Neosar, Neulasta,Neumega, Neupogen, Nilandron, Nilutamide, Nitrogen Mustard, Novaldex,Novantrone, Octreotide, Octreotide acetate, Oncospar, Oncovin, Ontak,Onxal, Oprevelkin, Orapred, Orasone, Oxaliplatin, Paclitaxel,Pamidronate, Panretin, Paraplatin, Pediapred, PEG Interferon,Pegaspargase, Pegfilgrastim, PEG-INTRON, PEG-L-asparaginase,Phenylalanine Mustard, Platinol, PlatinolAQ, Prednisolone, Prednisone,Prelone, Procarbazine, PROCRIT, Proleukin, Prolifeprospan 20 withCarmustine implant, Purinethol, Raloxifene, Rheumatrex, Rituxan,Rituximab, Roveron-A (interferon alfa-2a), Rubex, Rubidomycinhydrochloride, Sandostatin, Sandostatin LAR, Sargramostim, Solu-Cortef,Solu-Medrol, STI-571, Streptozocin, Tamoxifen, Targretin, Taxol,Taxotere, Temodar, Temozolomide, Teniposide, TESPA, Thalidomide,Thalomid, TheraCys, Thioguanine, Thioguanine Tabloid, Thiophosphoamide,Thioplex, Thiotepa, TICE, Toposar, Topotecan, Toremifene, Trastuzumab,Tretinoin, Trexall, Trisenox, TSPA, VCR, Velban, Velcade, VePesid,Vesanoid, Viadur, Vinblastine, Vinblastine Sulfate, Vincasar Pfs,Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VP-16, Vumon,Xeloda, Zanosar, Zevalin, Zinecard, Zoladex, Zoledronic acid, Zometa,Gliadel wafer, Glivec, GM-CSF, Goserelin, granulocyte colony stimulatingfactor, Halotestin, Herceptin, Hexadrol, Hexalen, Hexamethylmelamine,HMM, Hycamtin, Hydrea, Hydrocort Acetate, Hydrocortisone, Hydrocortisonesodium phosphate, Hydrocortisone sodium succinate, Hydrocortonephosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan, Idamycin,Idarubicin, Ifex, IFN-alpha, Ifosfamide, IL 2, IL-11, Imatinib mesylate,Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEGconjugate), Interleukin 2, Interleukin-11, Intron A (interferonalfa-2b), Leucovorin, Leukeran, Leukine, Leuprolide, Leurocristine,Leustatin, Liposomal Ara-C, Liquid Pred, Lomustine, LPAM, L-Sarcolysin,Meticorten, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol, MTC, MTX,Mustargen, Mustine, Mutamycin, Myleran, Iressa, Irinotecan,Isotretinoin, Kidrolase, Lanacort, L-asparaginase, and LCR. Theadditional anti-cancer agent can also include biopharmaceuticals suchas, for example, antibodies.

Many tumors and cancers have viral genome present in the tumor or cancercells. For example, Epstein-Barr Virus (EBV) is associated with a numberof mammalian malignancies. The compounds disclosed herein can also beused alone or in combination with anticancer or antiviral agents, suchas ganciclovir, azidothymidine (AZT), lamivudine (3TC), etc., to treatpatients infected with a virus that can cause cellular transformationand/or to treat patients having a tumor or cancer that is associatedwith the presence of viral genome in the cells. The compounds disclosedherein can also be used in combination with viral based treatments ofoncologic disease.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of cancer), duringearly onset (e.g., upon initial signs and symptoms of cancer), or afteran established development of cancer. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the chemopreventative treatment of subjects presenting precancerouslesions, those diagnosed with early stage malignancies, and forsubgroups with susceptibilities (e.g., family, racial, and/oroccupational) to particular cancers. Therapeutic treatment involvesadministering to a subject a therapeutically effective amount of thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein after cancer is diagnosed.

Also disclosed herein are methods of treating or preventing a disorderin a subject, such as a human, comprising administering to the subjectan effective amount of a compound disclosed herein or a pharmaceuticallyacceptable salt thereof. In some examples, the subject is an animal,such as a human. In some examples, the subject is identified as having aneed for treatment of the disorder. In some examples, the method treatsa disorder. In some examples, the disorder is associated withTNF-α-induced cell death, such as dysfunctional regulation ofTNF-α-induced cell death. In some examples, the disorder is associatedwith uncontrolled cellular proliferation, such as cancer. In someexamples, the disorder is cancer. In some examples, the disorder is aninflammatory disorder. In some examples, the disorder is an autoimmunedisorder, such as a disorder selected from rheumatoid arthritis,ankylosing spondylitis, Crohn's disease, psoriasis, hidradenitissuppurativa, and refractory asthma.

In some examples, the subject has been diagnosed with the disorder priorto the administration step.

In some examples, the compound is administered in an amount betweenabout 0.01 to 500 mg per kg patient body weight per day and can beadministered in single or multiple doses. In some examples, the dosagelevel can be about 0.1 to about 250 mg/kg per day, such as about 0.05 to100 mg/kg per day, or about 0.1 to 50 mg/kg per day. In some examples,the dosage can be 0.05 to 0.5, 0.5 to 5.0 or 5.0 to 50 mg/kg per day. Insome examples, the dosage level can be 0.5 to 100 mg/kg per day. Fororal administration, the compositions are can be provided in the form oftablets containing 1.0 to 1000 milligrams of the active ingredient, suchas 1.0, 5.0, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500,600, 750, 800, 900 and 1000 milligrams of the active ingredient for thesymptomatic adjustment of the dosage of the patient to be treated. Thecompound can be administered on a regimen of 1 to 4 times per day, suchas once or twice per day. This dosing regimen can be adjusted to providethe optimal therapeutic response.

In some examples, the subject is a domesticated animal. In someexamples, the domesticated animal is a domesticated fish, domesticatedcrustacean, or domesticated mollusk. In some examples, the domesticatedanimal is poultry. In some examples, the poultry is selected fromchicken, turkey, duck, and goose. In some examples, the domesticatedanimal is livestock. In some examples, the livestock animal is selectedfrom pig, cow, horse, goat, bison, and sheep.

In some examples, the method further comprises the step of identifyingthe animal in need of treatment or prevention of cancer. In someexamples, the mammal has been diagnosed with a need for treatment andprevention of cancer prior to the administering step.

Protection Against TNF-α Induced Cell Death

Also disclosed herein are methods for protection against TNFα-inducedcell death. The method can comprise administering an effective amount ofa compound disclosed herein, a compound made by a method disclosedherein, a library disclosed herein, or a compound identified by methodsdisclosed herein to a subject identified as having a need for protectionagainst TNFα-induced cell death.

In some examples, the amount is therapeutically effective. In someexamples, the amount is prophylactically effective.

In some examples, the subject is a cell. In some examples, the subjectis an animal. In some examples, the subject is a human.

Manufacture of a Medicament

Also disclosed herein are methods for the manufacture of a medicamentfor treating or preventing cancer comprising combining a therapeuticallyeffective amount of a disclosed compound or product of a disclosedmethod with a pharmaceutically acceptable carrier or diluent.

Also disclosed herein are methods for manufacturing a medicamentassociated with treating or preventing cancer or the need to treat orprevent cancer with a pharmaceutically acceptable carrier or diluent.

In some examples, the medicament comprises a disclosed compound.

Kits

Also disclosed are kits that comprise a compound disclosed herein in oneor more containers. The disclosed kits can optionally includepharmaceutically acceptable carriers and/or diluents. In one embodiment,a kit includes one or more other components, adjuncts, or adjuvants asdescribed herein. In another embodiment, a kit includes one or moreanti-cancer agents, such as those agents described herein. In oneembodiment, a kit includes instructions or packaging materials thatdescribe how to administer a compound or composition of the kit.Containers of the kit can be of any suitable material, e.g., glass,plastic, metal, etc., and of any suitable size, shape, or configuration.In one embodiment, a compound and/or agent disclosed herein is providedin the kit as a solid, such as a tablet, pill, or powder form. Inanother embodiment, a compound and/or agent disclosed herein is providedin the kit as a liquid or solution. In one embodiment, the kit comprisesan ampoule or syringe containing a compound and/or agent disclosedherein in liquid or solution form.

Also disclosed herein are kits comprising one or more of the disclosedcompounds, and one or more of: a) at least one anticancer compound, b)instructions for treating a disorder associated with cancer, or c)instructions for treating cancer.

In some examples, the kit further comprises at least one agent, whereinthe compound and the agent are co-formulated.

In some examples, the compound and the agent are co-packaged. The agentcan be any agent as disclosed herein, known to have a side effect ofcancer, an agent known to increase the risk of cancer, agent known totreat cancer in a subject.

The kits can also comprise compounds and/or products co-packaged,co-formulated, and/or co-delivered with other components. For example, adrug manufacturer, a drug reseller, a physician, a compounding shop, ora pharmacist can provide a kit comprising a disclosed compound and/orproduct and another component for delivery to a patient.

It is contemplated that the disclosed kits can be used in connectionwith the disclosed methods of making, the disclosed methods of using,and/or the disclosed compositions.

Method of Making Compounds

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions can vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on the compounds described herein include the addition,subtraction, or movement of the various constituents as described foreach compound. Similarly, when one or more chiral centers are present ina molecule, the chirality of the molecule can be changed. Additionally,compound synthesis can involve the protection and deprotection ofvarious chemical groups. The use of protection and deprotection, and theselection of appropriate protecting groups can be determined by oneskilled in the art. The chemistry of protecting groups can be found, forexample, in Wuts and Greene, Protective Groups in Organic Synthesis, 4thEd., Wiley & Sons, 2006, which is incorporated herein by reference inits entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

The disclosed compounds can be prepared by solid phase peptide synthesiswherein the amino acid α-N-terminal is protected by an acid or baseprotecting group. Such protecting groups should have the properties ofbeing stable to the conditions of peptide linkage formation while beingreadily removable without destruction of the growing peptide chain orracemization of any of the chiral centers contained therein. Suitableprotecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc),t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz),biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the disclosed compounds. Other preferredside chain protecting groups are, for side chain amino groups likelysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc),nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc, andadamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl,2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyland acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; forhistidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl;for tryptophan, formyl; for asparticacid and glutamic acid, benzyl andt-butyl and for cysteine, triphenylmethyl (trityl). In the solid phasepeptide synthesis method, the α-C-terminal amino acid is attached to asuitable solid support or resin. Suitable solid supports useful for theabove synthesis are those materials which are inert to the reagents andreaction conditions of the stepwise condensation-deprotection reactions,as well as being insoluble in the media used. Solid supports forsynthesis of α-C-terminal carboxy peptides is4-hydroxymethylphenoxymethyl-copoly(styrene-1% divinylbenzene) or4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° C. and 50° C. in a solvent such as dichloromethane or DMF. When thesolid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.One method for coupling to the deprotected 4(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin isO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer. In one example, the α-N-terminal inthe amino acids of the growing peptide chain are protected with Fmoc.The removal of the Fmoc protecting group from the α-N-terminal side ofthe growing peptide is accomplished by treatment with a secondary amine,preferably piperidine. Each protected amino acid is then introduced inabout 3-fold molar excess, and the coupling is preferably carried out inDMF. The coupling agent can beO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.). At the endof the solid phase synthesis, the polypeptide is removed from the resinand deprotected, either in successively or in a single operation.Removal of the polypeptide and deprotection can be accomplished in asingle operation by treating the resin-bound polypeptide with a cleavagereagent comprising thianisole, water, ethanedithiol and trifluoroaceticacid. In cases wherein the α-C-terminal of the polypeptide is analkylamide, the resin is cleaved by aminolysis with an alkylamine.Alternatively, the peptide can be removed by transesterification, e.g.with methanol, followed by aminolysis or by direct transamidation. Theprotected peptide can be purified at this point or taken to the nextstep directly. The removal of the side chain protecting groups can beaccomplished using the cleavage cocktail described above. The fullydeprotected peptide can be purified by a sequence of chromatographicsteps employing any or all of the following types: ion exchange on aweakly basic resin (acetate form); hydrophobic adsorption chromatographyon underivitized polystyrene-divinylbenzene (for example, AmberliteXAD); silica gel adsorption chromatography; ion exchange chromatographyon carboxymethylcellulose; partition chromatography, e.g. on SephadexG-25, LH-20 or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on octyl- oroctadecylsilyl-silica bonded phase column packing.

Disclosed herein are methods for making a bicyclic peptide compound, themethods can comprise: a) linking 2,3-diaminopropanoic acid to a solidphase support via its carboxyl functionality; b) building a firstpeptide chain of 2-6 amino acid residues from the 2-amino functionalityof the 2,3-diaminopropanoic acid residue; c) linking a lysine residue tothe distal end of the first peptide chain; d) building a second peptidechain of 3-7 amino acid residues onto the lysine residue; e) linkingtrimesic acid to the 3-amino functionality of the 2,3-diaminopropanoicacid residue; f) cyclizing the distal amino acid residue of the secondpeptide chain with a carboxyl functionality of the trimesic acid; and g)cyclizing the amino side chain of the lysine residue with a carboxylfunctionality of the trimesic acid.

In some examples, the method further comprises the step of linking alabel moiety to the compound.

In some examples, the method further comprises the step of cleaving thecompound from the solid phase support. Also disclosed is a method formaking a library of bicyclic peptide compounds, the method comprisingthe steps of: a) linking 2,3-diaminopropanoic acid to a solid phasesupport via its carboxyl functionality; b) building a first peptidechain of 2-6 amino acid residues from the 2-amino functionality of the2,3-diaminopropanoic acid residue, using a split-and-pool technique toprepare the chain; c) linking a lysine residue to the distal end of thefirst peptide chain; d) building a second peptide chain of 3-7 aminoacid residues onto the lysine residue, using a split-and-pool techniqueto prepare the chain; e) linking trimesic acid to the 3-aminofunctionality of the 2,3-diaminopropanoic acid residue; f) cyclizing thedistal amino acid residue of the second peptide chain with a carboxylfunctionality of the trimesic acid; and g) cyclizing the amino sidechain of the lysine residue with a carboxyl functionality of thetrimesic acid.

Methods of Identifying

Also disclosed herein are methods of identifying a drug candidate. Insome examples, the drug candidate is a compound disclosed herein or apharmaceutically acceptable salt thereof

Disclosed herein is a method for identifying a drug candidate fortreatment of a disorder, the method comprising the steps of: exposing acompound disclosed herein, a compound prepared by the methods disclosedherein, a library disclosed herein, or a library prepared by the methodsdisclosed to a receptor associated with the disorder; b) detectingreaction between the receptor and the compound or the library; and c)determining the identity of compound reacting with the receptor.

Also disclosed herein is a compound identified by the method of foridentifying a drug candidate for treatment of a disorder.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary of theinvention and are not intended to limit the scope of what the inventorsregard as their invention. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.), but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric.

Certain materials, reagents and kits were obtained from specific vendorsas indicated below, and as appropriate the vendor catalog, part or othernumber specifying the item are indicated.

Example 1

Fmoc-protected L-amino acids were purchased from Advanced ChemTech(Louisville, Ky.), Peptides International (Louisville, Ky.), or Aapptec(Louisville, Ky.). O-Benzotriazole-N,N,N,N′-tetramethyluroniumhexafluorophosphate (HBTU) and 1-hydroxybenzotriazole hydrate (HOBt)were from Aapptec. Tetramethylrhodamine azide was prepared as previouslydescribed (Wells, J and McClendon, C. Nature, 2007, 450, 1001-1009). Allsolvents and other chemical reagents were obtained from Sigma-Aldrich,Fisher Scientific (Pittsburgh, Pa.), or VWR (West Chester, Pa.) and wereused without further purification unless noted otherwise.N-(9-Fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) was fromAdvanced ChemTech. Phenyl isothiocyanate was purchased in 1-mL sealedampoules from Sigma-Aldrich, and a freshly opened ampoule was used ineach experiment.

Synthesis of Diallyl Trimesic Acid. Trimesic acid (2 g, 9.52 mmol) wasdissolved in 20 ml of allyl alcohol and cooled to 0° C. Five equiv. ofthionyl chloride and 0.1 equiv of DMF were slowly added to the abovesolution and the reaction mixture was allowed to warm to roomtemperature. The reaction was refluxed overnight and stopped byevaporation to dryness under reduced pressure. The residue was dissolvedin DCM and washed with saturated NaHCO₃ solution and brine. The organiclayer was dried over MgSO₄ and evaporated. The resulting product wasdissolved in 1:1 THF/allyl alcohol and 0.9 equiv. of KOH was added. Thesolution was stirred for 1 h. The reaction mixture was evaporated andthe residue was dissolved in DCM and extracted with 0.1 M NaOH. Theorganic layer was discarded. Concentrated HCl was added to the aqueouslayer until the product completely precipitated out of the solution. Theprecipitate was collected by vacuum filtration and dried under vacuum toafford the desired product (80% yield). ¹H-NMR (250 MHz, DMSO-d6): 68.67-8.69 (m, 3H), 6.02-6.18 (m, 2H), 5.47 (d, J_(irans)=17.5 Hz, 2H),5.35 (d, Jcis=10 Hz, 2H), and 4.88 (d, 4H).

Protein Expression and Purification. The gene coding for theextracellular domain of human TNFα (aa 77-233) was amplified by thepolymerase chain reaction using the full-length TNFα cDNA (OpenBiosystem) as template and oligonucleotides5′-catctcgagtcacagggcaatgatcccaaagt-3′ (SEQ ID NO.: 64) and5′-caccgcaagcttgtcagatcatcttctcgaacc-3′ (SEQ ID NO.: 65) as primers. Theresulting DNA fragment was digested with endonucleases Hind III and XhoI and inserted into prokaryotic vector pET22b(+)-ybbR (Yin, J. et al.Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 15815-15820). This cloningprocedure resulted in the addition of a ybbR tag (MVLDSLEFIASKL; SEQ IDNO.: 66) to the N-terminus of TNFα. E. coli BL21(DE3) cells transformedwith the pET22b-ybbR-TNFα plasmid were grown at 37° C. in Luria broth(LB) supplemented with 0.05 mg/ml ampicillin to an OD₆₀₀ of 0.50, whenprotein expression was induced by the addition of isopropylβ-D-1-thiogalactopyranoside (150 M final concentration). After 5 h at30° C., the cells (1 L) were harvested by centrifugation. The cellpellet was suspended in 20 mL of lysis buffer (40 mM Tris-HCl, 150 mMNaCl, pH 8.0) plus 0.5% protamine sulfate, 20 mg/mL trypsin inhibitor,100 mg/ml phenylmethylsulfonyl fluoride and 100 mg/mL lysozyme. Themixture was stirred at 4° C. for 30 min and briefly sonicated (2×10 spulses). The crude lysate was centrifuged to yield a clear supernatant,which was diluted 10 times in running buffer (20 mM Tris-HCl, 1 mM EDTA,0.5% triton X100, pH 8.0) and passed through Q-Sepherase column. Thecolumn was eluted with 50 mL of running buffer with a gradient of 0-1000mM NaCl. The ybbR tagged TNFα fractions were pooled and concentrated to˜1 mL using an Amicon Ultra-15 cellulose membrane filter. The resultingsolution was passed through a Mono-Q 10/100 GL anion-exchange columnequilibrated in the running buffer. The column was eluted with therunning buffer plus a linear gradient of 0-1000 mM NaCl. Fractionscontaining TNFα were pooled and concentrated in an Amicon Ultra-15cellulose filter. Protein concentration was determined by Bradford assayusing bovine serum albumin as the standard.

Protein labeling. Biotinylation of TNFα was carried out by treating theybbR-tagged TNFα protein (80 μM) in 50 mM HEPES, pH 7.4, 10 mM MgCl₂with Sfp phosphopantetheinyl transferase (1 μM) and biotin-CoA (Yin, J.et al. Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 15815-15820) (100 μM)for 30 min at room temperature. Texas-Red labeling of TNFα was similarlycarried out except that a Texas Red-CoA adduct (Yin, J. et al. Proc.Natl. Acad. Sci. U. S. A., 2005, 102, 15815-15820) was used instead ofbiotin-CoA. The reaction mixture was passed through a G-25Fast-Desalting column equilibrated in 30 mM HEPES, pH 7.4, 150 mM NaClto remove any free biotin or dye molecules.

Fluorescence Anisotropy. A primary FA experiment was performed byincubating 100 nM TMR-labeled bicyclic peptide with 2 μM TNFα in theblocking buffer. The full FA titration experiment was similarlyperformed by incubating 50 nM labeled bicyclic peptide with varyingconcentrations (0-6 μM) of TNFα. The FA values were measured on aMolecular Devices Spectramax M5 spectrofluorimeter, with excitation andemission wavelengths at 545 and 585 nm, respectively. Equilibriumdissociation constants (K_(D)) were determined by plotting thefluorescence anisotropy values as a function of TNFα concentration. Thetitration curves were fitted to the following equation

$Y = \frac{\begin{pmatrix}{A_{\min} +} \\{\left( {{A_{\max} \times \frac{Q_{b}}{Q_{f}}} - A_{\min}} \right)\left( \frac{\left( {L + x + K_{D}} \right) - \sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}}{2L} \right)}\end{pmatrix}}{\left( {1 + {\left( {\frac{Q_{b}}{Q_{f}} - 1} \right)\left( \frac{\left( {L + x + K_{D}} \right) - \sqrt{\left( {\left( {L + x + K_{D}} \right)^{2} - {4{Lx}}} \right)}}{2L} \right)}} \right)}$

where Y is the measured anisotropy at a given TNFα concentration x; L isthe bicyclic peptide concentration; Qb/Qf is the correction fact fordye-protein interaction; A_(max) is the maximum anisotropy when all thepeptides are bound to TNFα, while A_(min) is the minimum anisotropy.

Peptide Sequencing by PED-MS. Beads containing the encoding linearpeptides were placed into individual wells of an AcroPrep 96-well filterplate (Pall Corporation, PN5030) with one bead per well. To each wellwas added a freshly mixed solution containing 25 μL of pyridine/water(v/v 2:1) plus 0.1% triethylamine and 25 μL of Fmoc-OSu (2 μmol) andphenyl isothiocyanate (100 μmol) in dry pyridine. The reaction wasallowed to proceed for 6 min and drained by a universal vacuum manifoldsystem designed for 96-well plates (United Chemical Technologies, Inc.).The bead was washed five times with DCM and once with TFA, and incubatedwith 100 μL of TFA (2×6 min). The bead was washed with DCM and pyridineand PED cycle was repeated for 11 times. After the last PED cycle, theN-terminal Fmoc group was removed by treatment with 20% piperidine inDMF. For MALDI-TOF analysis, each bead was treated with 100 μL of TFAcontaining ammonium iodide (1.0 mg) and dimethylsulfide (10 μL) for 20min to reduce any oxidized Met. The bead was washed with water andtransferred into a microcentrifuge tube and treated overnight with 20 μLof CNBr in 70% TFA (40 mg/mL) in the dark. The solvents were evaporatedunder vacuum to dryness and the peptides released from the bead weredissolved in 5 μL of 0.1% TFA in water. One L of the peptide solutionwas mixed with 2 μL of saturated 4-hydroxy-α-cyanocinnamic acid inacetonitrile/0.1% TFA (1:1) and 1 μL of the mixture was spotted onto aMALDI sample plate. Mass spectrometry was performed on a BrukerMicroflex MALDI-TOF instrument. The data obtained were analyzed byMoverz software (Proteometrics LLC, Winnipeg, Canada).

Library Synthesis. The bicyclic peptide library was synthesized on 2.0 gof TentaGel S NH₂ resin (90 m, 0.2 mmol/g). All of the manipulationswere performed at room temperature unless otherwise noted. The linkersequence (BBFM) was synthesized with 4 equiv of Fmoc-amino acids, usingHBTU/HOBt/DIPEA as the coupling reagents. The coupling reaction wastypically allowed to proceed for 1 h, and the beads were washed with DMF(3×) and DCM (3×). The Fmoc group was removed by treatment twice with20% piperidine in DMF (5+15 min), and the beads were exhaustively washedwith DMF (6×). To spatially segregate the beads into outer and innerlayers, the resin (after removal of N-terminal Fmoc group) was washedwith DMF and water, and soaked in water overnight. The resin was quicklydrained and suspended in a solution of Fmoc-OSu (0.26 mmol, 0.50 equiv)and diisopropylethylamine (1.2 mmol or 2.0 equiv) in 30 mL of 55:45(v/v) DCM/diethyl ether. The mixture was incubated on a carousel shakerfor 30 min. The beads were washed with 55:45 DCM/diethyl ether (3×) andDMF (8×) to remove water from the beads and then treated with 5 equiv ofdi-t-butyl dicarbonate in DMF. Next, the Fmoc group was removed bypiperidine treatment and 2 equiv of 4-hydroxymethylbenzoic acid andHBTU/HOBt/DIEA (2:2:4 equiv) were added to the resin. Fmoc-β-Ala-OH (5equiv) was coupled to the Hmb linker by using DIC/DMAP (5.5:0.1 equiv),and the coupling was repeated twice to drive the reaction to completion.Then, Fmoc-L-Pra-OH, two Fmoc-(3-Ala-OH, and Fmoc-L-Dap(Alloc)-OH weresequentially coupled by standard Fmoc/HBTU chemistry. The Boc protectinggroup on the encoding peptide was removed by treatment withTFA/water/triisopropylsilane (95:2.5:2.5), and the exposed amine wascoupled with Fmoc-Arg(Pbf)-OH. The random region was synthesized by thesplit-and-pool method (Lam, K S et al. Nature, 1991, 354, 82-84;Houghten, R A et al. Nature, 1991, 354, 84-86; Furka, A et al. Int. J.Pep. Prot. Res., 1991, 37, 487-493; Thakkar, A et al. ACS Comb. Sci.,2013, 15, 120-129; Yin, J. et al. Proc. Natl. Acad. Sci. USA, 2005, 102,15815-15820) using 5 equiv of Fmoc-amino acids and HATU as the couplingagent. The coupling reaction was repeated once to ensure completereaction at each step. To differentiate isobaric amino acids duringPED-MS analysis, 4% (mol/mol) of CD₃CO₂D was added to the couplingreactions of D-Ala, D-Leu, D-Lys, and Om, while 4% CH₃CD₂CO₂D was addedto the Nle reactions (Thakkar, A et al. Anal. Chem., 2006, 78,5935-5939). Fmoc-Lys(Mmt)-OH was placed in the middle of the randompositions using HATU/DIPEA (4 and 8 equiv) to facilitate the formationof bicyclic compounds. After the entire sequence was synthesized, theAlloc group on the C-terminal Dap residue was removed by treatment witha DCM solution containing tetrakis(triphenylphosphine)palladium (0.25equiv) and phenylsilane (5 equiv) for 15 min (3×). The beads weresequentially washed with 0.5% diisopropylethylamine in DMF, 0.5% sodiumdimethyldithiocarbamate hydrate in DMF, DMF (3×), DCM (3×), and DMF(3×). The resulting free amine was coupled to diallyl protected trimesicacid using HATU/DIPEA (5 equiv, 10 equiv) for 2 h. The allyl protectinggroups on trimesic acid scaffold was removed using the same procedure asdescribed for the Alloc group. The lysine Mmt group was removed using 2%TFA/5% triisopropylsilane in DCM for 40 min. The N-terminal Fmoc groupwas then removed with 20% piperidine in DMF. The beads were washed withDMF (6×), 1 M HOBt in DMF (3×), DMF (3×), and DCM (3×). For peptidecyclization, a solution of PyBOP/HOBt/NMM (5, 5, 10 equiv, respectively)in DMF was mixed with the resin and the mixture was incubated on acarousel shaker for 3 h. The resin was washed with DMF (3×) and DCM (3×)and dried under vacuum for >1 h. Side chain deprotection was carried outwith modified reagent K (7.5% phenol, 5% water, 5% thioanisole, 2.5%ethanedithiol, 1% anisole, and 1% triisopropylsilane in TFA) for 1 h.The resin was washed with TFA and DCM and dried under vacuum beforestorage at −20° C.

Library Screening. Library resin (100 mg) was swollen in DCM, washedextensively with DMF, doubly distilled H₂O, and incubated in 1 mL ofblocking buffer (PBS, pH 7.4, 150 mM NaCl, 0.05% Tween 20 and 0.1%gelatin) containing 800 nM biotinylated TNFα overnight at 4° C. Thebeads were washed with the blocking buffer, suspended in 1 mL of theblocking buffer supplemented with 10 μL of M280 streptavidin-coatedDynabeads (Invitrogen), and incubated for 1 h at 4° C. The magneticbeads were separated from the rest of the resin using a TA Dynal MPC-1magnetic particle concentrator (Invitrogen). The hits from magneticscreening were transferred into a BioSpin column (0.8 mL, BioRad) andwashed exhaustively with 6 M guanidine hydrochloride, water, and theblocking buffer to remove the bound proteins. The second round ofscreening was performed by incubating the initial hits with 1.5 Mbiotinylated TNFα as described above. After washing with the blockingbuffer, the beads were suspended in 1 mL of the blocking buffercontaining streptavidin-alkaline phosphatase (1 g/mL finalconcentration) at 4° C. for 10 min. The beads were quickly washed with 1mL of the blocking buffer (3×) and 1 mL of a staining buffer (30 mMTris, pH 8.5, 100 mM NaCl, 5 mM MgCl₂, 20 μM ZnCl₂) (3×). Next, 1 mL ofthe staining buffer and 100 μL of a BCIP stock solution (5 mg/mL) wereadded to the beads and intense turquoise color developed on positivebeads in 25 min. The turquoise colored beads were manually removed undera dissecting microscope, and subjected to a third round of screeningafter extensive washing with PBS, ddH₂O, and 8 M guanidinehydrochloride. The resulting beads were incubated overnight at 4° C.with 300 nM Texas-red labeled TNFα in the blocking buffer. The beadswere viewed under an Olympus SZX12 microscope equipped with afluorescence illuminator (Olympus America, Center Valley, Pa.) and theintensely fluorescent beads were manually collected as positive hits.

On-Bead Labeling and Peptide Release: The positive beads derived fromon-bead screening were pooled, washed with water and DMF, and soaked in60 μL of 1:1 (v/v) water/DMF mixture. The labeling reaction wasinitiated by the addition of 20 μL of freshly prepared ascorbic acid andcopper sulfate solutions (each at 5 mg/mL in water) and 5 μL oftetramethylrhodamine azide in DMSO (10 mM). The reaction was allowed toproceed at room temperature overnight in the dark. The reaction wasterminated by extensive washing of the beads with water/DMF, and thebeads were transferred into individual microcentrifuge tubes (onebead/tube) and each treated with 5 μL of 0.1 M NaOH solution for 4 h atroom temperature in the dark. The solution was neutralized by theaddition of 5.5 μL of 0.1 M HCl, transferred to a new tube, evaporatedto dryness in a vacuum concentrator, and redissolved in 26 μL of doubledistilled water to generate a stock solution of ˜1 μM bicyclic peptide.The beads containing the linear coding peptides were kept in theoriginal tubes and stored for later PED-MS analysis.

Effect of Bicyclic Peptide on TNFα-TNRF1 Interaction. Recombinant TNFR1was purchased from R&D Systems. EZ-Link Plus activated peroxidase, anamine-reactive form of HRP was purchased from Thermo Scientific. TNFR1was labeled with HRP by combining 50 μL of TNFR1 (1.0 μM) and 4 μL ofHRP (2.7 pNI) in 950 pt, of Na₂CO₃ buffer (0.2 M Na₂CO₃, 0.15 M NaCl, pH9.0) for 1 h. The resulting TNFR1-HRP conjugate was treated with NaCNBH₃to reduce the resulting Schiff base and quenched with 20 μl ofethanolamine. A Nunc 96F Maxisorp plate was coated overnight with 100 μlof 5 mg/mL Neutravidin (in 50 mM Na₂CO₃, pH 9.0) at 4° C. The solutionwas removed and each well was washed with 100 μL of the blocking buffercontaining 3% BSA. Next, 100 μL of 7.5 nM biotinylated TNFα in PBS wasadded to each well and incubated at 4° C. for 1 h. The wells werequickly washed twice with a washing buffer (0.01% Tween 20 in PBSbuffer). Peptides of varying concentrations (50 μL) were added to thewells, followed by the addition of 50 μL of 0.5 nM TNFR1-HRP. Afterincubation at 4° C. for 1.5 h, the plate was washed twice with thewashing buffer and incubated with 100 μL of3,3′,5,5′-tetramethylbenzidine (Sigma) for 30 min. The reaction wasquenched by the addition of 100 μL of 1 M phosphoric acid. Theabsorbance at 450 nm was measured and plotted against the peptideconcentration and the IC₅₀ value was obtained by curve fitting.

MTT Assay. WEHI-13VAR fibroblasts (American Type Culture Collection)were seeded at a density of 5×10⁴ cells/well in 100 μL of culture medium(10% FBS in RPMI 1640) and allowed to grow overnight at 37° C. and 5%CO₂. TNFα (0.04 ng/mL final concentration), varying concentrations ofpeptide (0-25 μM), and actinomycin D (1 μg/mL) were mixed and incubatedfor 1 h in the CO₂ incubator. Next, 50 μl of the resulting mixture wasadded into each well and the plate was incubated overnight. Ten L of theMTT labeling reagent (final concentration 0.5 mg/ml) was added to eachwell and incubated for 4 h. One hundred L of the MTT solubilizationsolution (Roche) was added to each well and the plate was let stand inthe incubator overnight and the absorbance at 550 nm was measured andplotted as a function of the peptide concentration.

Bicyclic Peptidyl Antagonists of Tumor Necrosis Factor-Alpha

Design and Synthesis of Bicyclic Peptide Library. Antibodies recognizespecific antigens by utilizing six small loops, called the“complementarity determining regions”. By grafting two or more flexibleloops onto protein scaffolds, other investigators have engineeredprotein binders of antibody-like affinity and specificity (Koide, A etal. J. Mol. Biol., 1998, 284, 1141-1151; Beste, G et al. Proc. Natl.Acad. Sci. USA, 1999, 96, 1898-1903; Xu, L. H. et al. Chem. Biol., 2002,9, 933-942; Rutledge, S E et al. J. Am. Chem. Soc., 2003, 125,14336-14347; Steiner, D et al. J. Mol. Biol., 2008, 382, 1211-1227).Displaying peptidic or peptidomimetic loops on small-molecule scaffoldscan also generate molecules that rival antibodies for binding affinityand specificity. To develop inhibitors against PPIs, a planar structurewas chosen as the scaffold, in order to maximize the surface area of theresulting molecules and therefore their ability to interact with flatprotein surfaces. To test the validity of this approach, a bicyclicpeptide library was designed by “wrapping” a peptide sequence of 6-10random residues around a trimesoyl group (FIG. 1). Peptide cyclizationwas mediated by the formation of three amide bonds between trimesic acidand the N-terminal amine, the side chain of a C-terminalL-2,3-diaminopropionic acid (Dap), and the side chain of a fixed lysinewithin the random region. The resulting bicyclic peptides contained 3-5random residues in each ring. The random sequence was constructed with a25-amino acid set selected based on their structural diversity,metabolic stability, and commercial availability. It included 10proteinogenic α-L-amino acids [Ala, Arg, Asp, Gln, Gly, His, Ile, Ser,Trp, and Tyr], 5 nonproteinogenic α-L-amino acids[L-4-fluorophenylalanine (Fpa), L-norleucine (Nle), L-omithine (Orn),and L-phenylglycine (Phg)], and 10 a-D-amino acids [D-2-naphthylalanine(D-Nal), D-Ala, D-Asn, D-Glu, D-Leu, D-Lys, D-Phe, D-Pro, D-Thr, andD-Val]. This library has a theoretical diversity of 1.0×10¹⁴. Inpractice, the library size is limited by the amount of resin employedand typically on the order of 10⁷ (vide infra). Although the actualdiversity represents only a very small fraction of all possiblestructures, the library compounds can sample a large structural space.Once an active compound is identified, its affinity and specificity forthe desired target may be improved by synthesizing and screening asecond-generation library containing the analogs of the initial hit. Tomaximize the structural space while keeping the molecular weight to aminimum (e.g., <2000), it was concluded that in addition to the 20proteinogenic amino acids, unnatural amino acids and potentiallynonpeptidic building blocks can also be employed and such compoundlibraries can be most conveniently prepared by chemical synthesis.

The main challenge associated with screening chemically synthesizedbicyclic peptide libraries is structural determination of the hitcompounds; no methodology is yet available to directly sequence bicyclicpeptides derived from combinatorial libraries. To overcome thisdifficulty, the bicyclic peptide library was synthesized in the onebead-two compound format (Joo, S H et al. J. Am. Chem. Soc., 2006, 128,13000-13009; Liu, T et al. ACS Comb. Sci., 2011, 13, 537-546) on 2.0 gof TentaGel microbeads (90 μm, 2.86×10⁶ beads/g, ˜100 pmolpeptide/bead). Each library bead was topologically segregated into twodifferent layers, with the outer layer displaying a unique bicyclicpeptide and the inner layer containing the corresponding linear peptideas an encoding tag. To spatially segregate the beads, the TentaGel resinwas soaked in water, drained, and quickly suspended in 1:1 (v/v)DCM/Et₂O containing 0.5 equivalent ofN-(9-fluorenylmethoxycarbonyloxy)succinimide (Fmoc-OSu) (Liu, R et al.J. Am. Chem. Soc., 2002, 124, 7678-7680). Because the organic solvent isimmiscible with water, only peptides on the bead surface were exposed toand reacted with Fmoc-OSu. The beads were washed with DMF and theremaining free N-terminal amines inside the beads were protected with aBoc group. After removal of the Fmoc group, a p-hydroxymethylbenzoicacid (Hmb) linker was added (for selective release of the bicyclicpeptide), followed by the addition of β-Ala, L-propargylglycine (Pra),two β-Ala, and Fmoc-L-Dap(Alloc)-OH. The Pra residue serves as a handlefor selective labeling of the bicyclic peptide via click chemistry (videinfra). The Dap residue permits attachment of the bicyclic peptide tothe solid support as well as providing a side chain for peptidecyclization. The N-terminal Boc group was then removed from the innerpeptides by treatment with trifluoroacetic acid (TFA) and an arginineresidue was added to provide a fixed positive charge, which facilitateslater peptide sequencing by mass spectrometry. The random region wassynthesized by the split-and-pool method (Lam, K S et al. Nature, 1991,354, 82-84; Houghten, R A et al. Nature, 1991, 354, 84-86; Furka, A etal. Int. J. Pep. Prot. Res., 1991, 37, 487-493) and anNE-4-methoxytrityl (Mmt)-protected lysine was added in the middle of therandom sequence to provide a side-chain amine for peptide cyclization.Following completion of the linear peptide synthesis, the Mmt group wasremoved using 2% TFA in DCM and replaced with an Fmoc group (the Mmtgroup was partially removed during deprotection of the Alloc group). TheAlloc group on the C-terminal Dap was removed by treatment withPd(PPh₃)₄ and the exposed side chain amine was acylated with diallyltrimesic acid. Finally, the allyl (on the trimesoyl moiety) and Fmocprotecting groups (on the N-terminus and the lysine side chain) wereremoved and the surface peptides were cyclized by treatment withbenzotriazol-1-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate(PyBOP). The peptides inside the beads were unaffected by thecyclization procedure due to lack of the Dap residue and remained in thelinear form to serve as encoding tags. Note that macromolecular targets(e.g., proteins) cannot diffuse into the bead interior and thus thelinear encoding peptides do not interfere with library screening. Thesymmetry of the trimesoyl unit ensured that a single bicyclic productwas formed on each bead.

To assess the quality of the bicyclic library, 10 beads were randomlyselected for MS analysis. The peptide on each bead was released by CNBrcleavage after the C-terminal methionine and analyzed by MALDI-TOF MS.For each of the 10 beads, the released peptides showed two peaksseparated by 528 mass units, as expected for the molecular massdifference between the linear encoding sequence (m/z M) and thecorresponding bicyclic peptide (m/z M+528) (FIG. 6). Previous studieshave shown that on-resin cyclization of hexa- and longer peptides withPyBOP is essentially quantitative for ?99.96% of the sequences (Joo, S Het al. J. Am. Chem. Soc., 2006, 128, 13000-13009; Thakkar, A et al. ACSComb. Sci., 2013, 15, 120-129).

Library Screening Against TNFα. The bicyclic peptide library wassubjected to four rounds of screening against TNFα. TNFα was expressedin Escherichia coli as a fusion to an N-terminal ybbR tag(MVLDSLEFIASKL; SEQ ID NO.: 66) and specifically labeled at the ybbR tagwith a biotin or fluorescent dye by using phosphopantetheinyltransferase Sfp (Yin, J. et al. Proc. Natl. Acad. Sci. USA, 2005, 102,15815-15820). During the first round, 100 mg of the bicyclic peptidelibrary (˜3×10⁵ beads) was incubated with biotinylated TNFα (0.8 μM) andstreptavidin-coated magnetic particles. The resulting magnetic(positive) beads (˜400 beads) were separated from the rest of librarybeads by applying a magnetic field to the wall of the container; thepositive beads were attracted to the wall, while the negative beadssettled to the bottom of the container (magnetic sortin) (Kodadek, T andBachhawat-Sikder, K. Mol. BioSyst., 2006, 2, 25-35; Hu, B H et al. Anal.Chem., 2007, 79, 7275-7285). The ˜400 beads were washed, incubated againwith the biotinylated TNFα (1.5 μM), and subjected to a second round ofscreening using an on-bead enzyme-linked assay and astreptavidin-alkaline phosphatase (SA-AP) conjugate (Sweeney, M C et al.Biochemistry, 2005, 44, 14932-14947). Binding of TNFα to a bead recruitsSA-AP to the bead surface and upon the addition of5-bromo-4-chloro-3-indolyl phosphate (BCIP), produces a turquoisecolored precipitate on that bead. This procedure resulted in 150intensely turquoise colored beads, which were manually isolated using amicropipette with the aid of a dissecting microscope and washedexhaustively to remove the bound proteins and dye molecules. During thethird round of screening, the 150 beads were incubated with Texas-redlabeled TNFα (0.3 μM) and the 44 most fluorescent beads were manuallyisolated under a fluorescence microscope.

Finally, the 44 hits derived from the above on-bead assays wereindividually tested for binding to TNFα in solution by fluorescencepolarization (FA) (fourth round) (Liu, T et al. ACS Comb. Sci., 2011,13, 537-546; Hintersteiner, M et al. Chem. Biol., 2009, 16, 724-735).Thus, the 44 beads were treated with tetramethylrhodamine (TMR) azide inthe presence of Cu(I), resulting in selective labeling of the bicyclicpeptides at the Pra residue (FIG. 2A). The beads were then placed intoindividual microcentrifuge tubes (1 bead/tube) and the TMR-labeledbicyclic peptide was released from each bead by treatment with 0.1 MNaOH, which selectively hydrolyzed the Hmb ester linkage associated withthe bicyclic peptide. After neutralization and evaporation of solvent,the released bicyclic peptide from each bead was dissolved in water togive a stock solution of ˜1 μM. FA measurements were also carried out intwo stages. During the initial stage, each of the 44 bicyclic peptides(100 nM) was incubated with a single concentration of TNFα (5 μM) andthe anisotropy increase relative to the control (no TNFα) was measured.Out of the 44 peptides, 12 showed an increase of >15% (FIG. 2B). These12 bicyclic peptides were further analyzed at varying concentrations ofTNFα (0-18 μM) to generate a full binding curve for each peptide, fromwhich a dissociation constant (K_(D)) was calculated. Complete bindingcurves were obtained for 6 of the 12 bicyclic peptides (bead No. 1, 16,22, 24, 36, and 41) and their KD values ranged from 0.8 to 7.8 μM (FIG.7). No significant TNFα binding was observed for the bicyclic peptidesreleased from the other 6 beads (No. 18, 19, 28, 29, 31, and 44). Next,for the 6 bicyclic peptides that showed significant TNFα binding, thecorresponding beads containing the linear encoding peptides wereretrieved from the microcentrifuge tubes and subjected to partial Edmandegradation-mass spectrometry (PED-MS) analysis (Thakkar, A et al. Anal.Chem., 2006, 78, 5935-5939). Two of the beads (hits No. 1 and 36)produced mass spectra of sufficient quality, from which unambiguous,complete sequences ofbicyclo(Phg-Tyr-D-Ala-Lys-Tyr-D-Phe-Gly-D-Lys-His-Dap; SEQ ID NO.: 67)and bicyclo(Ala-D-Phe-Trp-D-Thr-Gln-Lys-Nle-D-Leu-Ala-His-Dap; SEQ IDNO. 68) were derived (FIG. 3A and FIGS. 8A and 8B). These compounds arenamed as Necrostatin C1 and C2 thereafter, respectively.

The fact that only a relatively small number of the hits derived fromon-bead screening (6 out of 44 beads) had strong binding to TNFα insolution suggests that many of the initial hits were false positives orweak binders, a common problem associated with on-bead screening whichis likely caused by the high ligand densities on the beads (˜100 mM) andmulti-dentate interactions (i.e., simultaneous interaction of a singleTNFα molecule with two or more resin-bound bicyclic peptides) (Chen, Xet al. J. Comb. Chem., 2009, 11, 604-611). False negatives are alsopossible due to certain technical difficulties (e.g., poor aqueoussolubility, inefficient release from resin by 0.1 M NaOH due to strongbinding to the TentaGel resin, and/or strong binding of a bicyclicpeptide to bovine serum albumin which was present in all FA assays).This highlights the importance of this library design, which permitsselective release of the bicyclic peptide and therefore solution-phasebinding analysis and avoids the need to individually resynthesize all 44initial hits.

Binding Affinity and Specificity for TNFα. To confirm their bindingaffinity and specificity for TNFα, Necrostatin C1, C2, and the linearand monocyclic variants of Necrostatin C1 were resynthesized with afluorescein isothiocyanate (FITC) label attached to the Dap residue(through a lysine linker) (FIG. 9A), purified by HPLC, and assayedagainst TNFα by FA analysis. Necrostatin C1 and C2 bound to TNFα withK_(D) values of 0.45 and 1.9 M, respectively (FIG. 3B). These K_(D)values are somewhat different from those derived from the single-bead FAanalysis (FIG. 7), probably because of impurities present in the beadderived peptide samples and the lower-than-expected peptideconcentration that made the single-bead analysis results less reliable.The linear C1 variant exhibited only weak binding to TNFα (K_(D)>10 μM),whereas the monocyclic peptide showed no significant binding affinity(FIG. 9B).Bicyclo(Arg-Arg-Arg-Arg-Nal-Phe-Dap-Ser-D-Val-Pro-p-Tyr-His-Dap; SEQ IDNO.: 69), a control peptide arbitrarily selected from another library(unrelated to TNFα), was also tested and had no detectable binding toTNFα (FIG. 9B). These results demonstrate that both the peptide sequenceand the overall bicyclic structure can play a role in binding to thetarget protein. Presumably, the bicyclic scaffold can restrict thepeptide sequence into a conformation(s) that is energeticallyinaccessible by the linear or monocyclic peptide. To determine whetherNecrostatin C1 and C2 are specific ligands of TNFα, they were tested forbinding to five other proteins of diverse structures and functions,including bovine serum albumin (BSA), a glutathione-S-transferase-PLC7SH2 domain fusion (GST-SH2), protein phosphatase PTP1B, HIV capsidprotein, and a GST-BRCT domain fusion protein. Necrostatin C1 showedweak binding to BSA (K_(D)˜34 μM), but not to any of the other proteins,while Necrostatin C2 was less selective showing varying affinities toBSA, GST-SH2, and PTP1B proteins (K_(D)=3.0-37 μM; FIGS. 10A and 10B).Finally, Necrostatin C2 (unlabeled) inhibited the binding ofFITC-labeled Necrostatin C1 and C2 to TNFα in a concentration-dependentmanner (IC₅₀ values of ˜4 and ˜2 μM, respectively) (FIG. 11), suggestingthat both compounds bind to the same (or overlapping) site on TNFα. Itshould be noted that intermediate binding affinity to serum proteins maybe of therapeutic benefits, as it greatly increases the residence timeof the therapeutic agent in circulation (Liu, X et al. Curr. Top. Med.Chem., 2011, 11, 450-466). Because of its higher affinity andspecificity for TNFα, Necrostatin C1 was selected for further biologicaltests.

Inhibition of TNFα-TNF Receptor Interaction by Necrostatin C1. TNFαsignaling begins with the binding of the TNFα trimer to theextracellular domain of TNFR1, triggering the release of the inhibitoryprotein, silencer of death domains (SODD), from the intracellular domainof TNFR1 (Chen, G and Goeddel, D V. Science, 2002, 296, 1634-1635). Totest whether Necrostatin C1 inhibits the interaction between TNFα andTNFR1, biotinylated TNFα was immobilized onto a Neutravidin-coated96-well microtiter plate. The plate was incubated with 0.5 nM horseradish peroxidase (HRP)-conjugated TNFR1 in the presence of varyingconcentrations of Necrostatin C1. After washing, the amount of HRP-TNFR1bound to each well was quantitated by ELISA using3,3′,5,5′-tetramethylbenzidine (TMB) as HRP substrate, which wasconverted into 3,3′,5,5′-tetramethylbenzidine diimine by HRP resultingin absorbance at 450 nm (Martin, T L et al. J. Histochem. Cytochem.,1984, 32, 793). Necrostatin C1 inhibited the TNFα-TNFR1 interaction in aconcentration-dependent manner, with an ICso value of 3.1±0.3 μM (FIG.4).

Necrostatin C1 Protects Cells from TNFα-Induced Apoptosis. The abilityof Necrostatin C1 to protect cells against TNFα-induced cell death wastested with cultured WEHI-13VAR fibroblasts, which are highly sensitiveto TNFα in the presence of actinomycin-D (LD₅₀ range: 0.005-0.065 ng/mL)(Khabar, K S et al. Immunol. Lett., 1995, 46, 107-110). WEHI-13VAR cellswere treated with a fixed concentration of TNFα (0.04 ng/ml) and varyingconcentrations of Necrostatin C1 (0-25 μM) and the fraction of livecells was quantitated by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay. Necrostatin C1 protected the cells from TNFα-induced cell deathin a concentration-dependent manner, whereas the correspondingmonocyclic and linear peptides did not (FIG. 5). The control bicyclicpeptide, bicycle(Arg-Arg-Arg-Arg-Nal-Phe-Dap-Ser-D-Val-Prop-Tyr-His-Dap;SEQ ID NO.: 70), which does not bind TNFα, had no protective effect. TheMTT assay was also conducted at a fixed concentration of Nercostatin C1(50 μM) but varying concentrations of TNFα (0-250 ng/mL). Under theassay conditions, TNFα exhibited an LD₅₀ value of 0.46 ng/mL in theabsence of TNFα inhibitor; in the presence of 50 μM Necrostatin C1, theLD₅₀ value was shifted to 1.8 ng/mL (FIG. 12). Taken together, this datasuggest that Necrostatin C1 binds to TNFα at or near the TNFα-TNFR1interface.

Conclusions. In conclusion, disclosed herein is a methodology forchemical synthesis and screening of large combinatorial libraries ofbicyclic peptides against macromolecular targets of biomedicalsignificance. Compared to the previously reported methods for bicyclicpeptide library synthesis (Sun, Y et al. Org. Lett., 2001, 3, 1681-1684;Virta, P and Lonnberg, H J. J Org. Chem., 2003, 68, 8534; Hennis, C etal. Nat. Chem. Biol., 2009, 5, 502-507; Chen, S et al. ChemBioChem.,2012, 13, 1032-1038; Sako, Y et al. J. Am. Chem. Soc., 2008, 130,7232-7234; Timmerman, P et al. J Biol. Chem., 2009, 284, 34126-34134),most of which involve ribosomal peptide synthesis followed by chemicalcyclization, the disclosed methods have the advantage that it allows theincorporation of any unnatural amino acid or non-peptidic buildingblocks, resulting in greater structural diversity and improved metabolicstability of the cyclic peptides. In addition, chemical synthesis allowsfor the use of orthogonal protecting groups, which in turn permits more“forcing” reaction conditions to drive the desired cyclization reactionto completion and can prevent any undesired cyclization reaction fromoccurring. It was also demonstrated that bicyclic peptides, such as thecompounds disclosed herein, containing a planar scaffold are capable ofbinding to flat protein surfaces such as PPI interfaces. With a K_(D)value of 0.45 μM, Necrostatin C1 is the most potent non-protein TNFαinhibitor reported to date. The bicyclic library methodology describedhere should be applicable to other proteins and nucleic acid targets.

Example 2

Bicyclic Peptide Ligands Against K-Ras

Approximately 300 mg of the bicyclic library was screened againstbiotinylated or fluorescently labeled K-Ras in 4 rounds, as describedfor TNFα. This screening produced 130 initial hits after the third round(screening against Texas red-labeled K-Ras). FA analysis at a singleconcentration of peptide (˜100 nM) and K-Ras protein (5 mM) showed that8 hits produced FA increases of ≥25%. These 8 hits were subjected tofull binding curve analysis using 50 nM peptide and 0-20 μM K-Ras. Sixshowed significant binding in the solution phase and their correspondingbeads were sequenced by PED-MS. Complete sequences were obtained forhits #38, #28, #13, #82, and #105 (Table 4). Resynthesized hit #13showed no significant binding while resynthesized 28 showed weak binding(K_(D)>5 μM).

TABLE 4 Binding affinities of hits from library. Binding affinity fromK_(D) of single- resyn- SEQ bead thesized Hit ID analysis peptide No.Sequence^(a) NO. (K_(D), μM) (μM)  13 Gln-Gln-val-Asp-Lys-Fpa- 94  5.1  ± NA phe-nal-ala-Gly-Dap   1.8NA  28 Tyr-nal-leu-Lys-ala-Gln- 95  3.2 ±  6.8 ± Ala-Gly-Ser-Dap   1.6  4.5  38 Trp-phe-Asp-Lys-phe-asn-71   2.6 ± 0.49 ± His-Dap   0.6 0.08  82 nal-Ser-Gln-nal-Phg-Lys- 72  3.3 ±  2.1 ± phe-Arg-val-Arg-Dap   1.1  0.9 105AArg-Arg-nal-Arg-Fpa-Lys- 73 ND  1.4 ± phe-glu-Gly-Dap  0.4 105BOrn-Arg-nal-Arg-Fpa-Lys- 74 0.052 ±  2.6 ± phe-glu-Gly-Dap 0.020  1.3^(a)The three-letter codes for L-amino acids have the first lettercapitalized, whereas those of D-amino acids have all lower-case letters.NA, no significant binding activity; ND, not determined.

Bicyclic peptides 13, 28, 38, 82, and 105 were resynthesized, labeledwith fluorescein isothiocyanate (FITC) at an added C-terminal Lys (FIG.22a and Scheme 1), and analyzed for binding to K-Ras by FA.

Peptides 28, 38, 82 and 105 bound to recombinant K-Ras with K_(D) valuesof 6.3, 0.49, 2.1, and 2.6 μM (FIG. 22b and Table 4) and were named as“cyclorasin B1-4” (for cyclic ras inhibitor bicyclic), respectively.Peptide 13 did not show significant binding to K-Ras. The discrepancybetween the binding affinities derived from single-bead analysis andthose determined with resynthesized and purified peptides may be causedby impurities present in the peptide samples released from the singlebeads (e.g., truncated peptides), which may interfere with the bindingof bicyclic peptide with K-Ras. Cyclorasin B2-4 were selected forfurther characterization because of their relatively high potencies.

The ability of cyclorasin B2-4 to block Ras-effector interactions wasfirst evaluated by a qualitative bead-binding assay (Wu, et al., Med.Chem. Commun. 2013, 4, 378-382). Briefly, GST-Raf was immobilized onglutathione beads and incubated with Texas red-labeled Ras protein;binding of the Ras protein to the immobilized Raf rendered the beadsintensely red (FIG. 23a ). However, the Ras-Raf interaction wascompletely abolished in the presence of 10 M cyclorasin B3 or B4 (FIG.23b ), but not cyclorasin B2. The potency for inhibition of the Ras-Rafinteraction was next determined by a homogeneous time resolvedfluorescence (HTRF) assay (Leyris, J. P. et al. Anal. Biochem. 2011,408, 253-262), giving an IC₅₀ value of ˜1.4 μM for cyclorasin B3 (FIG.23c ). Cyclorasin B2 again showed no inhibition. The HTRF assay failedfor cyclorasin B4 due to aggregation and precipitation of B4 at higherconcentrations. The ability of cyclorasin B2-4 to compete with theRas-binding domain (RBD) of Raf (GST-Raf RBD) and compound 12, themonocyclic K-Ras inhibitor we previously reported, was examined forbinding to K-Ras using an FA-based competition assay. Addition ofGST-Raf RBD (FIG. 23d ) or compound 12 (FIG. 23e ) inhibited the bindingof FITC-labeled cyclorasin B3 to K-Ras in a concentration-dependentmanner. Compound 12 also abolished the binding of cyclorasin B4 but notB2 to K-Ras. These results suggest that like compound 12, cyclorasin B3and B4 bind to a site(s) at or near the Ras-Raf interface, whereascyclorasin B2 binds to a site different from the Ras-Raf interface.

To assess the specificity of cyclorasin B2-4 for K-Ras, the ability ofcyclorasin B2-4 to self-compete for binding to K-Ras and to bind otherproteins was examined. Addition of unlabeled cyclorasin B2 inhibited thebinding of FITC-labeled B2 to K-Ras in a concentration-dependent manner.Likewise, unlabeled cyclorasin B4 inhibited the binding of FITC-labeledB4 to K-Ras. These results further support the notion that cyclorasinB2-4 each bind to a specific site on K-Ras. To determine whether thebicyclic structure is important for binding to K-Ras, the monocyclic andlinear counterparts of cyclorasin B2 were synthesized and their bindingaffinity for K-Ras was measured. The monocyclic and linear peptidesbound to K-Ras with K_(D) values of 7.4 and 58 μM, or 15- and 120-foldlower affinity than cyclorasin B2, respectively (FIG. 22b ). Therefore,both the bicyclic structure and the amino acid sequence are required forhigh-affinity binding to K-Ras. To test whether the cyclic peptides haveany selectivity for the signaling-active form of Ras, K-Ras wasspecifically loaded with GTP, GDP, or GPPNP (a GTP analog). CyclorasinB2 bound to all three Ras forms with essentially the same affinity(K_(D)=0.49, 0.64, and 0.76 μM, respectively) (FIG. 22d ). In contrast,cyclorasin B3 bound to Ras-GTP and Ras-GPPNP with approximately 8-foldhigher affinity than Ras-GDP (K_(D) values of 1.2, 1.6, and 9.3 μM,respectively) (FIG. 22e ). Finally cyclorasin B2 and B3 were tested forbinding to five arbitrarily selected proteins, including bovine serumalbumin (BSA), protein-tyrosine phosphatase 1B (PTP1B), GST-SHD SH2domain fusion protein, maltose-binding protein-XIAP BIR3 domain fusion(MBP-BIR3), and GST-FKBP12 fusion protein. Cyclorasin B2 is a selectiveK-Ras ligand, showing only weak binding to BSA, GST-SHD SH2, andMBP-BIR3 proteins (K_(D)=23-57 μM, which are 47-120-fold higher thanthat of K-Ras) but no binding to PTP1B or GST-FKBP12. Cyclorasin B3 issomewhat less selective than cyclorasin B2 and bound to MBP-BIR3 and BSAwith K_(D) values of 16 and 17 μM, respectively, and very weakly toPTP1B, GST-SHD SH2, and GST-FKBP12.

Cyclorasin B2-B4 were tested for inhibition of cell proliferation by theMTT assay. None of the compounds showed significant effect on theproliferation of cultured cancer cells up to 50 μM concentration, due topoor membrane permeability of the cyclic peptides (as determined byconfocal microscopy of cells treated with FITC-labeled peptides). Whenattached to an oleic acid group to improve its membrane permeability(Scheme II), cyclorasin B3 exhibited modest anti-proliferative activityagainst H358 lung cancer cells (FIG. 23f ).

Conjugation of cyclorasin B2 to a fatty acid or cell-penetrating peptide(Arg₁₁) failed to confer any cellular activity.

Example 3

Synthesis of Diallyl Nitrilotriacetic Acid

Synthesis of [2]. To a flame dried 25 mL round bottom flask was added 1(nitrilotriacetic acid, 1.00 g, 5.24 mmol, 1.0 eq.) and allyl alcohol(10 mL). Thionyl chloride (2 mL, 26.18 mmol, 5.0 eq.) was added dropwiseat 0° C., followed by DMF (40 μL, 0.524, 0.1 eq.). The reaction wasallowed to warm to room temperature before being heated at 75° C.overnight. The reaction was concentrated under reduced pressure, andwater (10 mL) and DCM (10 mL) were added. The organic layer was washedwith saturated sodium carbonate solution (1×), and then extracted withDCM (3×). The combined organic layers were dried (MgSO₄), filtered, andconcentrated to yield 2 quantitatively.

Synthesis of [3]. To crude residue of 2 was added THF (10 mL), KOH(0.264 g, 4.7 mmol, 0.9 eq.), and allyl alcohol (6 mL). The mixture wasallowed to stir for 1 h at room temperature, and then concentrated underreduced pressure. It was then extracted with isopropyl alcohol (3×), andthe extracts were combined, filtered, and concentrated to yield amixture of 2 and 3 (˜60% 3 by NMR).

Example 4

Peptide Library Design

A bicyclic peptide library (library I) was designed in the form ofFmoc-NH-Xs-Lys(Mtt)(Membrane transporter)-Dap(Aloc)-BBM-Resin (where Bis β-Alanine) utilizing a previously published strategy (FIG. 13). TheC-terminal ring contains a membrane transporter sequence in twodirections (Phe-Nal-Arg-Arg-Arg-Arg (SEQ ID NO: 96) orArg-Arg-Arg-Arg-Nal-Phe (SEQ ID NO: 97)) while the N-terminal ringcontains the random amino acids. To facilitate the attachment of a smallmolecule to the library through click chemistry, propargyl glycine (Pra)was incorporated in the random region. The amount of Pra on each beadwas reduced by 10-fold by co-coupling Fmoc-Pra-OH with an inert aminoacid (Fmoc-Lys(Ac)-OH) at a ratio of 10% Pra to 90% Lys(Ac). The Lys(Ac)residue conveniently serves as the encoder for sequencing after the Praresidue was modified by click chemistry. At each random position, 20% ofthe library was separated and coupled with Pra/Lys(Ac). The remaining80% of the library was split equally into 25 vessels and coupled with anamino acid from the set of 25 amino acids, which includes 10proteinogenic amino acid (Ala, Asp, Gly, His, Ile, Gln, Arg, Ser, Trp,Tyr), 9 D-amino acids (ala, glu, phe, lys, leu, asn, pro, thr, val), and6 unnatural amino acids (L-Abu (replaced Cys), L-Nle (replaced Met),L-Phg, L-Fpa, D-Nal, L-Orn) (FIG. 14).

The library was synthesized on 90 μm TentaGel resin, starting with theaddition of the BBM linker using standard Fmoc/HBTU chemistry. Spatialsegregation of the beads into two layers was achieved by soaking thebeads in water overnight, following by treating with 0.5 eq. of Fmoc-OSuin 1:1 (v/v) CH₂C12/Et₂O. The remaining (˜50%)N-terminal amines in thebead interior were capped by Boc anhydride. Subsequent removal of theFmoc group in the outer layer, following by coupling withFmoc-Dap(Aloc)-OH ensured only the outer layer can undergobi-cyclization. After the removal of the Boc and Fmoc groups, the beadswere split into two equal portions and the membrane transporter sequencewas coupled in two opposite directions (Phe-Nal-Arg-Arg-Arg-Arg (SEQ IDNO: 96) or Arg-Arg-Arg-Arg-Nal-Phe (SEQ ID NO: 97)). The beads werepooled and coupled with Fmoc-Lys(Mtt)-OH. At each random position, 20%of the beads were coupled with 9:1 Fmoc-Lys(Ac)-OH/Fmoc-Pra-OH while theremaining 80% were split and coupled with one of the 25 amino acids.

To facilitate the incorporation of small molecule and bi-cyclization,the Mtt protecting group was removed by 2% TFA in DCM, and the exposedLys side chain was protected by treatment with Fmoc-OSu. After removalof the Aloc group on the side chain of Dap, the beads were treated withthe appropriate azide (80-100 mM), 25 mM CuSO₄ and 25 mM ascorbic acidin 8:1:1 DMF/H₂O/t-butyl alcohol overnight (click chemistry). Afterwashing to remove the click chemistry reagents, trimesic acid wascoupled to the side chain of Dap using HATU as coupling reagent. Removalof the Fmoc groups with piperidine and treatment with pyBOP/HOBt/NMMafforded the bicyclic peptides containing the small-molecule “headgroup” on the Pra residues (FIG. 15).

Selection and synthesis of small molecule probe

Several small molecules have been reported to bind to K-Ras with lowaffinity. Among them, 4,6-dichloro-2-methyl-3-aminoethyl-indole (DCAI)is attractive due to its low molecular weight, relatively good bindingaffinity to K-Ras (1.1 mM) and commercial availability. The co-crystalstructure of DCAI with K-Ras revealed that the indole portion binds to apocket on the surface of K-Ras while the alkyl amine only has minimalinteraction with the protein. This provides a convenient position forattachment to bicyclic peptides through first conversion of the amine toazide and subsequent click chemistry with the alkyne group of Praresidues. DCAI was converted to the corresponding azide usingimidazole-1-sulfonyl azide hydrochloride as the diazo transfer reagent(FIG. 16).

Screening Strategy and Results

GST-tagged K-Ras G12V protein was purified and labeled with biotin. Atypical screening reaction involved incubating 100 mg of the libraryresin (˜300,000 beads) with the biotinylated K-Ras overnight. Afterwashing, the beads were be treated with streptavidin-alkalinephosphatase (SA-AP), followed by 5-Bromo-4-chloro-3-indolyl phosphate(BCIP). Binding of K-Ras to a bed recruits SA-AP to the beads (throughbinding to biotin by streptavidin). After dephosphorylation by alkalinephosphatase, the indolyl product dimerizes in air to form a turquoisecolor on positive beads. The colored beads were selected under amicroscope and subjected to sequencing by PED/MS.

Screening 100 mg of the DCAI-labeled bicyclic library againstbiotinylated K-Ras (300 nM) produced 21 hits, resulting in 13 fullsequences after PED/MS analysis (FIG. 20). The sequences revealedseveral trends. The transporter sequence is preferred in the forwardorientation (Phe-Nal-Arg-Arg-Arg-Arg (SEQ ID NO: 96)), with 12 out ofthe 13 hits containing this motif. The Pra/Lys(Ac) (Z) residue ispreferred at the X₁ or X₃ position, with all sequences containing Z ateither X₁, X₃ or both positions (FIG. 20). When a sequence contains twoZ residues, it is most likely that one of them is Pra while the other isLys(Ac).

Binding Analysis of Selected Peptide by Fluorescence Anisotropy

To confirm the screening results, four hit sequences (peptides A2, A8,B5 and B8; hereafter named as peptides 1-4) were selected forresynthesis and tested for binding to K-Ras in solution by fluorescenceanisotropy. The peptides were synthesized on Rink Amide resin, cleaved,deprotected, and purified by reversed-phase HPLC. Each peptide containsa Lys outside of the bicyclic rings to serve as the point of labelingwith an amine-reactive fluorescent dye (FAM-NHS or FITC). Peptides 2 and4 showed fairly potent binding to K-Ras, with K_(D) values of 5.1 μM and9 μM, respectively. Peptide 3 binds to K-Ras with weak affinity (20 μM),while peptide 1 does not bind to K-Ras (Table 5).

TABLE 5 Binding Affinities of peptides 1-4 against K-Ras. NB =no binding SEQ Pep- ID K_(D) tide Sequence NO. (μM) 1bicyclo(Arg-Asp-Phg-Pra-d-Asn-K- 75 NB FNalR₄-Dap)-K 2bicyclo(Phg-Arg-d-Asn-Pra-Ile-K- 76  5.1 FNalR₄-Dap)-K 3bicyclo(Pra-Ser-Phg-Lys(Ac)- 77 20 Lys(Ac)-K-FNalR₄-Dap)-K 4bicyclo(Pra-Arg-d-Val-Asp-Ala-K- 78  9 FNalR₄-Dap)-K

To test whether peptide 2 and DCAI bind to similar sites, a competitionexperiment was performed in which the binding of FITC-labeled peptide 2to K-Ras was assayed in the presence of increasing concentrations ofDCAI (FIG. 17). DCAI inhibited the binding of peptide 2 with an IC₅₀value of 1.4 mM, indicating that peptide 2 occupies the same bindingpocket that recognizes DCAI.

Optimization of Peptide 2

To test whether ring expansion would improve the binding affinity, fourpeptides (peptide 5-8) were synthesized by inserting one to three Alainto the Pra-containing ring. It was found that the addition of only oneAla to either side of the Pra-containing sequence (peptide 5 and 6) didnot significantly improve the binding (K_(D) of 6.4 μM and 5.6 μMrespectively). However, adding one Ala residue to both sides of themotif (peptide 7) improved the binding affinity to 1.8 μM while theaddition of three Ala residues (two to the N-terminus and one to theC-terminus of Pra motife) improved the binding affinity to 1.1 μM (Table6).

TABLE 6 Binding affinities of peptide 5-8 against K-Ras SEQ Pep- IDK_(D) tide Sequence NO. (μM) 5 bicyclo(Ala-Phg-Arg-d-Asn-Pra-Ile- 79 6.4K-FNalR₄-Dap)-K 6 bicyclo(Phg-Arg-d-Asn-Pra-Ile-Ala- 80 5.6K-FNalR₄-Dap)-K 7 bicyclo(Ala-Phg-Arg-d-Asn-Pra-Ile- 81 1.8Ala-K-FNalR₄-Dap)-K 8 bicyclo(Ala-Ala-Phg-Arg-d-Asn-Pra- 82 1.1Ile-Ala-K-FNalR₄-Dap)-K

It was next tested whether replacement of the added Ala residues byother amino acids might further improve the potency of the peptide. Afocused bicyclic library (library II) based on the sequence of peptide 2was synthesized in the form of:bicyclo(X¹-X²-Phg-Arg-d-Asn-Pra-Ile-X³-K-FNalR₄-Dap)-BBM-TentaGelbicyclo(X₁-X₂-Phg-Arg-d-Asn-Pra-Ile-X₃-K-FNalR₄-Dap)-BBMTentaGel (SEQ IDNO: 98, underlined portion), where X is any of the 25 amino acids usedfor library I. Screening 50 mg of library 2 against 100 nM ofbiotinylated K-Ras produced 25 hits, which gave 19 full sequences afterPED/MS. The results showed that small hydrophobic amino acids (Ala,d-Ala) are preferred at X¹ position while X² and X³ positions had littleselectivity (FIG. 21).

Nine of the hit peptides (D4, D6, D9, E1, E3, E4, E5, E6, and E8) wereresynthezided and tested for binding to K-Ras in solution. Peptidesselected from library II showed only moderately improved bindingaffinities over peptide 2, with K_(D) values of 1.1 μM-6 μM (Table 7).Peptide 11 [bicyclo(A-dL-Phg-R-dN-Pra-I-D-K-FNalR₄-Dap-K] showed thehighest affinity toward K-Ras (1.1 μM) and was chosen for furtheroptimization.

TABLE 7 Binding affinities of peptide selected fromlibrary II toward K-Ras. NT = not tested SEQ Pep- ID K_(D) tide SequenceNO. (μM)  9 bicyclo(d-Ala-Abu-Phg-Arg-d-Asn-Pra- 83 2.5Ile-Abu-K-FNalR₄-Dap)-K 10 bicyclo(Phg-Ile-Phg-Arg-d-Asn-Pra- 84 2.4Ile-Abu-K-FNalR₄-Dap)-K 11 bicyclo(Ala-d-Leu-Phg-Arg-d-Asn-Pra- 85 1.1Ile-Asp-K-FNalR₄-Dap)-K 12 bicyclo(d-Ala-Gln-Phg-Arg-d-Asn-Pra- 86 2Ile-Asp-K-FNalR₄-Dap)-K 13 bicyclo(Ala-Orn-Phg-Arg-d-Asn-Pra- 87 6.3Ile-d-Phe-K-FNalR₄-Dap)-K 14 bicyclo(d-Ala-Phg-Phg-Arg-d-Asn-Pra- 88 4.9Ile-d-Phe-K-FNalR₄-Dap)-K 18 bicyclo(d-Ala-Abu-Phg-Arg-d-Asn-Pra- 89 NTIle-Abu-K-FNalR₄-Dap)-K 19 bicyclo(Ala-Ala-Phg-Arg-d-Asn-Pra- 90 NTIle-Ala-K-FNalR₄-Dap)-K 20 bicyclo(Ala-Ala-Phg-Arg-d-Asn-Pra- 91 4Ile-Ala-K-FNalR₄-Dap)-K

Peptides 8 and 11 were subjected to a conventional medicinal chemistrySAR campaign, by making conservative substitutions (e.g., Ile to Leu,d-Asn to d-Asp). It was found that replacement of the Phg residue provedto be fruitful. For example, substitution of Phe for the Phg residues inpeptide 8 and 11, resulted in peptides 29 and 41, which have K_(D)values of 0.8 and 0.3 μM for K-Ras, respectively (Table 8).

TABLE 8 Binding affinities of peptide 29 and 41 SEQ Pep- ID K_(D) tideSequence NO. (μM) 29 bicyclo(Ala-Ala-Phe-Arg-d-Asn-Pra- 92 0.8Ile-Ala-K-FNalR₄-Dap)-K 41 bicyclo(Ala-d-Leu-Phe-Arg-d-Asn- 93 0.3Pra-Ile-Asp-K-FNalR₄-Dap)-K

Peptide 41 is Biologically Active

Peptide 41 was evaluated for anti-proliferative activity against H1299lung cancer cells by MTT proliferation assays. One hundred μL of H1299cells (0.5×10⁵ cells/mL) were placed in each well of a 96-well cultureplate and allowed to grow overnight. Varying concentrations of peptide41 (0-40 μM) were added to the each well and the cells were incubated at37° C. with 5% CO₂ for 24-72 h. Ten μL of a MTT stock solution (5 mg/ml)was added into each well. The plate was incubated at 37° C. for 4 h.Then 100 μL of SDS-HCl solubilizing buffer was added into each well, andthe resulting solution was mixed thoroughly. The plate was incubated at37° C. overnight. The absorbance of the formazan product was measured at570 nm using a Molecular Devices Spectramax M5 plate reader. Cellstreated with DMSO were used as control. Peptide 41 showed moderateanti-proliferative activity with an ED₅₀ of ˜9 μM (FIG. 18). Under thesame conditions, a control peptide which lacks the DCAI moiety but isotherwise identical to peptide 41 showed an ED₅₀>40 μM after the 24-hourtreatment. After 72 h, peptide 41 inhibited cell growth with an ED₅₀˜7μM. The control peptide also exhibited moderate cellular toxicity, butwas again less potent than peptide 41 (FIG. 19).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A compound of Formula I:

wherein: A is benzene; p, q, and r are independently selected from 0, 1,and 2; B₁, B₂, and B₃ are independently selected from O and NR¹; whereinR¹ is H, or substituted or unsubstituted C₁-C₅ alkyl; L₁ is selectedfrom substituted or unsubstituted C₁-C₆ alkyl, amino acid, and a linkerto a solid phase support; L₂ is selected from hydrogen, substituted orunsubstituted C₁-C₆ alkyl, amino acid, and a linker to a solid phasesupport; D is selected from substituted or unsubstituted C₁-C₆ alkyl, oramino acid residue; and X_(m) and X_(n) independently comprise asequence of 1-10 amino acids.
 2. The compound of claim 1, wherein one ormore of p, q, and r is
 1. 3. The compound of claim 1, wherein L₁ and L₂are independently selected from an amino acid.
 4. The compound of claim1, wherein L₁ is lysine.
 5. The compound of claim 1, wherein D is anamino acid residue.
 6. The compound of claim 1, wherein one or more ofB¹, B², and B³ is NR¹, and R¹ is H.
 7. The compound of claim 1, whereineach of p, q, and r is 0; L¹ is lysine; each of B¹, B², and B³ is NR¹,wherein R¹ is H; and D-L² is —CH₂—CH(NH)—C(O)—NH—R₀.
 8. The compound ofclaim 7, wherein the compound is of Formula I-A:

wherein: X_(m) and X_(n) are as defined in Formula I; R₀ is selectedfrom hydrogen, substituted or unsubstituted C₁-C₆ alkyl; and K has astructure represented by a formula:


9. The compound of claim 1, wherein X_(m) and X_(n) comprise differentamino acid sequences.
 10. The compound of claim 1, wherein X_(m)comprises 3-7 residues.
 11. The compound of claim 1, wherein X_(m)comprises 4-6 residues.
 12. The compound of claim 1, wherein X_(n)comprises 2-6 residues.
 13. The compound of claim 1, wherein X_(n)comprises 3-5 residues.
 14. The compound of claim 1, wherein thecompound has a molecular weight of 500-5000.
 15. The compound of claim1, wherein X_(m) and X_(n) are selected from SEQ ID NO: 1-4 and 6-63.16. The compound of claim 1, wherein X_(m) and X_(n) are selected fromSEQ ID NO: 1-4 and 6-25.